Localization and expression of molt-inhibiting hormone and nitric oxide synthase in the central nervous system of the green shore crab, Carcinus maenas, and the blackback land crab, Gecarcinus lateralis.

In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca2+-dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~105-fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iß) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca2+-dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt.

Increased Caspase-6 activity in the human anterior olfactory nuclei of the olfactory bulb is associated with cognitive impairment.

Abnormally elevated hippocampal Caspase-6 (Casp6) activity is intimately associated with age-related cognitive impairment in humans and in mice. In humans, these high levels of Casp6 activity are initially localized in the entorhinal cortex, the area of the brain first affected by the formation of neurofibrillary tangles, according to Braak staging. The reason for the high vulnerability of entorhinal cortex neurons to neurofibrillary tangle pathology and Casp6 activity is unknown. Casp6 activity is involved in axonal degeneration, therefore, one possibility to explain increased vulnerability of the entorhinal cortex neurons would be that the afferent neurons of the olfactory bulb, some of which project their axons to the entorhinal cortex, are equally degenerating. To examine this possibility, we examined the presence of Casp6 activity, neurofibrillary tangle formation and amyloid deposition by immunohistochemistry with neoepitope antisera against the p20 subunit of active Casp6 and Tau cleaved by Casp6 (Tau∆Casp6), phosphorylated Tau paired helical filament (PHF-1) antibodies and anti-ß-amyloid antiserum, respectively, in brains from individuals with no or mild cognitive impairment and Alzheimer disease (AD) dementia. Co-localization of Casp6 activity, PHF-1 and ß-amyloid was detected mostly in the anterior olfactory nucleus (AON) of the olfactory bulb. The levels of active Casp6 in the AON, which were the highest in the AD brains, correlated with PHF-1 levels, but not with ß-amyloid levels. AON Tau∆Casp6 levels correlated with entorhinal cortex Casp6 activity and PHF-1 levels. Multiple regression analyses demonstrated that AON Casp6 activity was associated with lower global cognitive function, mini mental state exam, episodic memory and semantic memory scores. These results suggest that AON Casp6 activity could lead to Casp6-mediated degeneration in the entorhinal cortex, but cannot exclude the possibilities that entorhinal cortex degeneration signals degeneration in the AON or that the pathologies occur in both regions independently. Nevertheless, AON Casp6 activity reflects that of the entorhinal cortex.

Activation of Glycogen Synthase Kinase-3 Mediates the Olfactory Deficit-Induced Hippocampal Impairments.

The populations with olfactory dysfunction show an increased chance for hippocampus-dependent episodic memory deficit. Although it is known that the olfactory information projects to the hippocampus through entorhinal cortex layer II, the molecular mechanisms linking olfactory deficit to the hippocampus is not understood. Using bilateral olfactory bulbectomy (OBX) as a model, we found that OBX induced memory deficits with activation of several memory-related protein kinases in the hippocampal extracts, including glycogen synthase kinase-3ß (GSK-3ß), protein kinase A (PKA), extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB). The OBX rats also show suppression of long-term potentiation (LTP); reduction of synapsin I, synaptophysin, NR2A/B, and PSD95; thinner presynaptic active zone and postsynaptic density with enlarged synaptic space; decreased spine numbers and mushroom-type spines; and tau hyperphosphorylation. After injection of SB216763 for several weeks by vena caudalis, selective inhibition of GSK-3ß ameliorated the OBX-induced memory deficits with recovery of the synaptic components and tau phosphorylation. Furthermore, genetic ablation of GSK-3ß by lentivirus-packed shRNA effectively rescued the memory deficits, synaptic disorder, and tauopathy. Our data indicate that GSK-3 activation mediates the olfactory deficits to the hippocampus, and targeting GSK-3 blocks the pathological connection.