Expression and alternative splicing of classical and nonclassical MHCI genes in the hippocampus and neuromuscular junction.

The major histocompatibility complex class I (MHCI) is a large gene family, with over 20 members in mouse. Some MHCIs are well-known for their critical roles in the immune response. Studies in mice which lack stable cell-surface expression of many MHCI proteins suggest that one or more MHCIs also play unexpected, essential roles in the establishment, function, and modification of neuronal synapses. However, there is little information about which genes mediate MHCI's effects in neurons. In this study, RT-PCR was used to simultaneously assess transcription of many MHCI genes in regions of the central and peripheral nervous system where MHCI has a known or suspected role. In the hippocampus, a part of the CNS where MHCI regulates synapse density, synaptic transmission, and plasticity, we found that more than a dozen MHCI genes are transcribed. Single-cell RT-PCR revealed that individual hippocampal neurons can express more than one MHCI gene, and that the MHCI gene expression profile of CA1 pyramidal neurons differs significantly from that of CA3 pyramidal neurons or granule cells of the dentate gyrus. MHCI gene expression was also assessed at the neuromuscular junction (NMJ), a part of the peripheral nervous system (PNS) where MHCI plays a role in developmental synapse elimination, aging-related synapse loss, and neuronal regeneration. Four MHCI genes are expressed at the NMJ at an age when synapse elimination is occurring in three different muscles. Several MHCI mRNA splice variants were detected in hippocampus, but not at the NMJ. Together, these results establish the first profile of MHCI gene expression at the developing NMJ, and demonstrate that MHCI gene expression is under tight spatial and temporal regulation in the nervous system. They also identify more than a dozen MHCIs that could play important roles in regulating synaptic transmission and plasticity in the central and peripheral nervous systems.

MHCI promotes developmental synapse elimination and aging-related synapse loss at the vertebrate neuromuscular junction.

Synapse elimination at the developing neuromuscular junction (NMJ) sculpts motor circuits, and synapse loss at the aging NMJ drives motor impairments that are a major cause of loss of independence in the elderly. Here we provide evidence that at the NMJ, both developmental synapse elimination and aging-related synapse loss are promoted by specific immune proteins, members of the major histocompatibility complex class I (MHCI). MHCI is expressed at the developing NMJ, and three different methods of reducing MHCI function all disrupt synapse elimination during the second postnatal week, leaving some muscle fibers multiply-innervated, despite otherwise outwardly normal synapse formation and maturation. Conversely, overexpressing MHCI modestly accelerates developmental synapse elimination. MHCI levels at the NMJ rise with aging, and reducing MHCI levels ameliorates muscle denervation in aged mice. These findings identify an unexpected role for MHCI in the elimination of neuromuscular synapses during development, and indicate that reducing MHCI levels can preserve youthful innervation of aging muscle.

Dominant negative actions of human prostacyclin receptor variant through dimerization: implications for cardiovascular disease.

OBJECTIVE: Prostacyclin and thromboxane mediate opposing cardiovascular effects through their receptors, the prostacyclin receptor (IP) and thromboxane receptor (TP). Individuals heterozygous for an IP variant, IP(R212C), displayed exaggerated loss of platelet IP responsiveness and accelerated cardiovascular disease. We examined association of IP(R212C) into homo- and heterodimeric receptor complexes and the impact on prostacyclin and thromboxane biology. METHODS AND RESULTS: Dimerization of the IP, IP(R212C), and TPalpha was examined by bioluminesence resonance energy transfer in transfected HEK293 cells. We observed an equal propensity for formation of IPIP homodimers and IPTPalpha heterodimers. Compared with the IP alone, IP(R212C) displayed reduced cAMP generation and increased endoplasmic reticulum localization but underwent normal homo- and heterodimerization. When the IP(R212C) and IP were coexpressed, a dominant negative action of the variant was evident with enhanced wild-type IP localization to the endoplasmic reticulum and reduced agonist-dependent signaling. Further, the TPalpha activation response, which was shifted from inositol phosphate to cAMP generation following IPTPalpha heterodimerization, was normalized when the TPalpha instead dimerized with IP(R212C). CONCLUSIONS: IP(R212C) exerts a dominant action on the wild-type IP and TPalpha through dimerization. This likely contributes to accelerated cardiovascular disease in individuals carrying 1 copy of the variant allele.

Searchable high-resolution 2D gel proteome of the human colon crypt.

We seek alterations in protein patterns at the earliest possible step on the path to cancer, namely, in cells of the target tissue from normal persons versus the corresponding normally appearing cells from persons who are heterozygous for mutation in a tumor suppressor gene that predisposes strongly to carcinoma in that tissue. To begin a systematic comparison of the proteomes of cells from normal and from neoplastic colons, we have undertaken the isolation of human colon crypts that are derived from the normal-appearing mucosa of left (descending) colon of patients with sporadic colorectal cancer. Two-dimensional (2D) gel electrophoresis is a proteomic approach that excels in the resolution of protein isoforms. Here, we document the practicality of this approach with human samples using gels of three overlapping pH ranges. For the first time, about 800 nonredundant proteins and 900 isoforms from purified human colonic crypts were identified, permitting an assessment of the contributions of protein isoforms. These interactive, searchable, hyperlink-enabled proteome maps and gene ontology analyses will facilitate future studies to discover the earliest markers and intervention targets during progression to colon cancer.