Macrophage-derived vascular endothelial growth factor-A is integral to neuromuscular junction reinnervation after nerve injury

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (Vegf-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and Vegf-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes

A novel carboxyl-terminal heptapeptide initiates the regulated secretion of LH from unique sub-domains of the ER.

The coordinated secretion of LH and FSH are critical for reproductive functions. After translocation into the endoplasmic reticulum (ER), their biosynthetic routes diverge at a determinative step prior to sorting in the regulated (LH) and constitutive (FSH) secretion pathways. Recently, we identified a C-terminal heptapeptide sequence, present only in the LHβ subunit, as a critical signal for entry of the LH dimer into the regulated pathway. We showed that an LHβ mutant lacking the heptapeptide (LHβΔT) assembled more efficiently with the α subunit than wild-type LHβ subunit, and this LHΔT dimer was secreted constitutively. Thus, an association exists between the presence of the C-terminal heptapeptide and sorting of the LH heterodimer to the regulated pathway. To study how this delayed LHβ subunit assembly is related to the trafficking of LH, we exploited the single subunit transfection model in rat somatotrope-derived GH3 cells with the use of immunofluorescence confocal microscopy. The LHβ subunit showed a distinct immunofluorescent localization as compared to the FSHβ subunit and LHβ mutants. The wild-type LHβ subunit exhibited a perinuclear staining corresponding to the ER/nuclear envelope region. In contrast, the wild-type FSHβ subunit and the mutants LHβΔT and LHβL119A displayed no detectable perinuclear staining; only peripheral ER puncta were observed. Also, no perinuclear fluorescence was detected in cells expressing the LH heterodimer. We propose that the C-terminal heptapeptide is responsible for delayed heterodimer assembly within an ER sub-domain of the nuclear envelope, as an early partitioning event necessary for the entrance of LH into the regulated secretory pathway, whereas FSHβ does not traverse the nuclear envelope region. These data suggest that, at least for LH, the molecular decision to enter the regulated secretory pathway is a pre-Golgi event controlled by the novel C-terminal heptapeptide

Summary of subunit/chaperone localization in the ER of GH₃ cells.

<p>Summary of subunit/chaperone localization in the ER of GH₃ cells.</p

Immunostaining of LHβ subunit in CHO (A) and MDCK (B) cells.

<p>The cells were immunoprobed with CGβ antiserum (<i>green</i>). <i>Note</i> that LHβ shows dispersed cytoplasmic puncta (A, B, <i>arrowhead</i>) with no ring-like pattern near nucleus. The n indicates the nucleus (<i>red</i>). The micrographs shown are representative of four experiments. X150.</p

Schematic diagram of human gonadotropin subunits.

<p>The crosshatched area of the region 115–121 denotes the heptapeptide of the LHβ subunit. N, Asn-linked oligosaccharides.</p

Representative Western blot of cell lysates (50 µg total protein/lane) derived from GH₃ cells.

<p>(<b>A</b>) The migration of subunits (<i>arrows</i>) and molecular mass markers are indicated. <i>Note</i> the longer time exposure (Exp.) for FSHβ and FSHβ-L (lanes 4 and 5) compared to LHβ and mutants (lanes 1–3). Bands at approximate 25 kDa presumably represents protein aggregates (*). In addition, LHβΔT and FSHβ-L are separated on SDS-PAGE gel into 2 bands (<i>arrows</i>). β-Actin was used as an internal control. (B). Histogram of densitometric measurements for LHβ, FSHβ and mutants. The protein level for LHβ and FSHβ was arbitrarily set as 1. Fold changes in expression level of LHβ mutants and FSHβ-L were compared with LHβ and FSHβ, respectively. Each value indicates the mean <b>±</b> SEM (n = 3). *Significant difference from LHβ with <i>p</i><0.05.</p

Subcellular localization of LHβΔT (A), FSHβ-L (B), LHβL119A (C) subunit and LH dimer (D) in GH₃ cells.

<p>The cells were immunostained with CGβ antiserum (<i>green</i>) and a monoclonal antibody against FSHβ subunit (<i>green</i>). <i>Note</i> unique ER perinuclear staining pattern for FSHβ-L mutant (B, <i>arrow</i>) vs. dispersed cytoplasmic puncta for LHβΔT and LHβL119A subunit (A, C, <i>arrowhead</i>) or LH dimer (D, <i>arrowhead</i>). The n indicates the nucleus (<i>red</i>). The micrographs shown are representative of four to eight experiments. IgG (E), mouse immunoglobulin. ×100.</p

Immunolocalization of endogenous BiP (A, B) and calnexin (CNX, C) in non-transfected GH₃ or CHO cells.

<p>For GH₃ cells the BiP antiserum (A, <i>red</i>) stained predominantly around nuclei (<i>arrow</i>), while the CNX antiserum (C, <i>red</i>) showed peripheral ER staining (<i>arrowhead). Note</i> that BiP in CHO cells (B) is localized as dispersed cytoplasmic puncta with some aggregation near the NE (<i>arrowhead</i>). Nuclei (n) were counterstained using TOPRO-iodide-3 (<i>blue</i>). The micrographs shown are representative of four experiments.</p