Gama-Enolase is a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C-terminal end of the molecule. We have investigated the expression and co-localization of Gama-enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of gama-enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C-terminally cleaved form of gama-enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of gama-enolase in microglial cells in response to amyloid-ß peptide (Aß) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with gama-enolase proved to be neuroprotective against Aß toxicity, however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of gama-enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid-ß-related neurodegeneration.
COBISS.SI-ID: 3441265
Aim To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimers disease pathogenesis and to define the cell types containing increased levels of Syt IV in the -amyloid plaque vicinity. Methods Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markersfor astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronalspecific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex. Results Western blot analysis showed higher Syt IV levelsin Tg2576 mice cortex than in WT cortex. Syt IV was foundonly in neurons. In plaque vicinity, Syt IV was up-regulatedin dystrophic neurons. The Syt IV signal was not up-regulatedin the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions). Conclusions Syt IV up-regulation within dystrophic neuronsprobably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimers diseaseand is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.
COBISS.SI-ID: 30928601
In rat fast muscles, collagen Q (ColQ) expression is restricted to the neuromuscular junctions. In contrast, it is high also extrajunctionally in the slow soleus muscles. Fast muscles activated by chronic low-frequency electrical stimulation, similar to neural activation of the soleus muscles, did not increase their extrajunctional expression of ColQ. We assumed that the myogenic stem cells (satellite cells) in fast and slow muscles were intrinsically different in regard to the capacity that they convey to their respective muscle fibers to increase the extrajunctional ColQ expression upon innervation. ColQ mRNA levels were determined by quantitative real-time PCR. Extensive neural suppression of the extrajunctional ColQ expression in regenerating fast muscles during maturation is a very slow process requiring 3060 days. If the immature regenerating fast EDL muscles were indirectly or directly electrically stimulated immediately after innervation by chronic low-frequency impulse pattern for 8 days, no significant increase of the extrajunctional ColQ mRNA levels was observed in stimulated regenerates in comparison to non-stimulated ones. In contrast, the extrajunctional ColQ mRNA levels in the regenerates of the soleus muscles, trans-innervated by the EDL nerve at the time of muscle injury, increased 4- to 5- fold after 8 days of the same chronic low-frequency electrical stimulation in comparison to those in the stimulated EDL regenerates. Since both fast and slow muscles completely regenerated only from their own myogenic stem cells and were innervated by the same nerve and later activated by the same tonic pattern of impulses, these results demonstrated that the mechanism causing incapacity of regenerating fast muscles to increase their extrajunctional ColQ expression upon tonic activation is encoded in their satellite cells, which in this respect differ from those in the slow muscles.
COBISS.SI-ID: 30454745
We developed a staining protocol that enables simultaneous visualization of myosin heavy chain (MHC) pure and hybrid muscle fiber types in rat skeletal muscle. Up to eight different muscle fiber types can be visualized in a single section of the rat extensor digitorum longus muscle, which contains all four adult MHC isoforms and shows plasticity during the denervation-reinnervation process. Triple immunofluorescent staining of MHC-1, MHC-2a and MHC-2b with primary antibodies BA-D5 (isotype IgG2b), SC-71 (isotype IgG1) and BF-F3 (isotype IgM) and with three fluorophore-labeled isotype-specific secondary antibodies displays different muscle fiber types in a merged image of red, green and blue channels, each in its own color. Immunoperoxidase staining with primary antibody 6H1 directed against MHC-2x can be additionally applied on the same tissue section to facilitate the identification of muscle fibers containing MHC-2x. Triple staining can also be used in combination with other staining procedures to derive more information about the number of capillaries or the oxidative potential of muscle fiber types. Simultaneous visualization of multiple fiber types in a single merged image enables economical use of muscle samples and provides simple and rapid identification of all fiber types that are present in rat limb muscles
COBISS.SI-ID: 30372825
Difference in phenotypes of sensory neurons innervating dental pulp or gingivomucosa may be responsible for intense pain sensations in pulpitis in contrast to relatively painless chronic periodontitis. Therefore, in a rat model, we classified these neurons according to their size and two neurochemical characteristics of nociceptors, their TrkA expression and isolectin IB4 binding. We conclude that the dental pulp and gingivomucosa are richly innervated by nociceptive TrkA-expressing neurons. However, while the great majority of pulpal neurons are larger NGF-dependent A-fibre nociceptors without affinity to bind IB4, almost half of the gingival neurons are smaller IB4 binding C-fibre nociceptors. The difference in phenotype of sensory neurons might partially explain the different sensitivity of both tissues during normal and pathological conditions.
COBISS.SI-ID: 512343609