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Sensitivity and reliability of animal behavioral assessment methods are critical for successful translation of in vitro findings to in vivo. Here we report a data transformation process in the elevated open platform task that generates a novel parameter, namely peak tolerance of fear (PTF) or its inversely correlated equivalent of anxiety quotient (AQ), to measure anxiogenic tendency in rodent. As compared to traditional parameters such as travel distance, time, or entries, PTF or AQ displays largely reduced data dispersion not only ingroup but also cross-study and cross-cohort, therefore representing a significant improvement of the methodology for rodent anxiety assessment.

Open field (OF) and elevated plus maze (EPM) have been widely used for assessing rodent innate fear. In OF task, animals with more fear will spend less time in the center zone (unsafe zone). Regarding EPM, animals with more fear will spent less time in the open arms (unsafe zone). Thus less activity in unsafe zone will be observed in animals with more fear. Based on this assumption, we speculated that our previously designed opaque round elevated platform (O-REP) with open space can be used for assessing rodent innate fear by analyzing animal activity. In O-REP, height and the open space make the animals feel fearful in O-REP and decrease its activity. To verify this novel method, the difference between female and male mice were examined by OF, EPM, and O-REP respectively. The results showed that EPM task could not find the significant fear difference between female and male mice using ICR and C57/BL6 strain. However, C57/BL6 female mice displayed less fear both in OF and O-REP task, which indicates that OF and O-REP task displayed more sensitivity than EPM task in assessing rodent innate fear. Furthermore, O-REP task rather than OF task found that female ICR mice displayed significantly more innate fear in O-REP, which indicated that O-REP might be more sensitive than OF and EPM in assessing innate fear. Further investigations showed that the animals displayed similar activities in O-REP, opaque square elevated platform (O-SEP) and transparent round elevated platform (T-REP), indicating that shape and transparency of EP did not affect the sensitivity of EP. Thus our data demonstrated that the REP is novel alternative task for evaluating rodent innate fear, which sensitivity was not affected by the shape and transparency. The present studies will greatly facilitate fear related research.

Obstructive sleep apnea (OSA) leads to cognitive impairment in about 25% patients, though it remains elusive what makes one more susceptible than the other to be cognitively impaired. G protein-coupled receptor kinase-5 (GRK5) deficiency is recently found to render subjects more susceptible to cognitive impairment triggered by over-expression of Swedish mutant ß-amyloid precursor protein. This study is to determine whether GRK5 deficiency also renders subjects more susceptible to the OSA-triggered cognitive impairment. Both wild type (WT) and GRK5 knockout (KO) mice were placed in conditions absence and presence of intermittent hypoxia (IH) with 8%/21% O2 90-second cycle for 8 hours a day for a month, and then followed by behavioral assessments with battery of tasks. We found that the selected IH condition only induced marginally abnormal behavior (slightly elevated anxiety with most others unchanged) in the WT mice but it caused significantly more behavioral deficits in the KO mice, ranging from elevated anxiety, impaired balancing coordination, and impaired short-term spatial memory. These results suggest that GRK5 deficiency indeed makes the mice more susceptible to wide range of behavioral impairments, including cognitive impairments.

G protein-coupled receptors (GPCRs) mediate a wide variety of physiological function. GPCR signaling is negatively regulated by the receptor desensitization, a procedure initiated by a group of kinases, including GPCR kinases (GRKs). Studies using genetargeted mice revealed that deficiency of a particular GRK member led to dysfunction of a highly selective group of GPCRs. In particular, for example, GRK5 deficiency specifically disrupts M2/M4-mediated muscarinic cholinergic function. Emerging evidence indicates that ?-amyloid accumulation may lead to GRK5 deficiency, while the latter impairs desensitization of M2/M4 receptors. Within memory circuits, M2 is primarily presynaptic autoreceptor serving as a negative feedback to inhibit acetylcholine release. The impaired desensitization of M2 receptor by GRK5 deficiency leads to hyperactive M2, which eventually suppresses acetylcholine release and results in an overall cholinergic hypofunctioning. Since the cholinergic hypofunctioning is known to cause ?-amyloid accumulation, the GRK5 deficiency appears to connect the cholinergic hypofunctioning and ?-amyloid accumulation together into a self-amplifying cycle, which accelerates both changes. Given that the ? -amyloid accumulation and the cholinergic hypofucntioning are the hallmark changes in the ?-amyloid hypothesis and the cholinergic hypothesis, respectively, the GRK5 deficiency appears to bring the two major hypotheses in Alzheimer’s disease together, whereas the GRK5 deficiency is the pivotal link. Therefore, any strategies that can break this cycle would be therapeutically beneficial for Alzheimer’s patients.

Why certain diseases primarily affect one specific neuronal subtype rather than another is a puzzle whose solution underlies the development of specific therapies. Selective basal forebrain cholinergic (BFC) neurodegeneration participates in cognitive impairment in Alzheimer’s disease (AD), yet the underlying mechanism remains elusive. Here, we report the first recapitulation of the selective BFC neuronal loss that is typical of human AD in a mouse model termed GAP. We created GAP mice by crossing Tg2576 mice that over-express the Swedish mutant human β-amyloid precursor protein gene with G protein-coupled receptor kinase-5 (GRK5) knockout mice. This doubly defective mouse displayed significant BFC neuronal loss at 18 months of age, which was not observed in either of the singly defective parent strains or in the wild type. Along with other supporting evidence, we propose that GRK5 deficiency selectively renders BFC neurons more vulnerable to degeneration.

In recent years, occurrence of "general paresis (GP)" has increased significantly because of the increasing incidence of syphilis in China. Early diagnosis plays a very important role for effective treatment. Incidence is becoming extensive enough to warrant an updated investigation of the clinical characteristics of GP. The authors retrospectively reviewed 116 cases of GP in Guangzhou, China, and analyzed its incidence and clinical appearance, as well as the characteristics of EEG, neuroradiology, serum, and cerebrospinal fluid examinations. Of the 116 GP patients, clinical symptoms presented frequently on admission were a variety of psychiatric-behavioral symptoms and varying degrees of dementia. Positive sucking reflex was the most common sign, as well as hyperreflexia and Argyll-Robertson pupil. EEG data mainly showed slightly abnormal EEG activity, with increased δ waves. Focal atrophy in one or multiple cerebral regions was evident on neuroimage. The prevalence of GP extends to various social strata or classes, with clinical presentation varying considerably among patients. For patients with progressive cognitive and behavioral deterioration, accompanied with psychotic and/or affective behavioral disorders or cerebral atrophy of unknown cause, general paresis should be considered.

Membrane G protein-coupled receptor kinase 5 (GRK5) deficiency is linked to Alzheimer disease, yet its precise roles in the disease pathogenesis remain to be delineated. We have previously demonstrated that GRK5 deficiency selectively impairs desensitization of presynaptic M2 autoreceptors, which causes presynaptic M2 hyperactivity and inhibits acetylcholine release. Here we report that inactivation of one copy of Grk5 gene in transgenic mice overexpressing β-amyloid precursor protein (APP) carrying Swedish mutations (Tg2576 or APPsw) resulted in significantly increased β-amyloid (Aβ) accumulation, including increased Aβ(+) plaque burdens and soluble Aβ in brain lysates and interstitial fluid (ISF). In addition, secreted β-APP fragment (sAPPβ) also increased, whereas full-length APP level did not change, suggesting an alteration in favor of β-amyloidogenic APP processing in these animals. Reversely, perfusion of methoctramine, a selective M2 antagonist, fully corrected the difference between the control and GRK5-deficient APPsw mice for ISF Aβ. In contrast, a cholinesterase inhibitor, eserine, although significantly decreasing the ISF Aβ in both control and GRK5-deficient APPsw mice, failed to correct the difference between them. However, combining eserine with methoctramine additively reduced the ISF Aβ further in both animals. Altogether, these findings indicate that GRK5 deficiency accelerates β-amyloidogenic APP processing and Aβ accumulation in APPsw mice via impaired cholinergic activity and that presynaptic M2 hyperactivity is the specific target for eliminating the pathologic impact of GRK5 deficiency. Moreover, a combination of an M2 antagonist and a cholinesterase inhibitor may reach the maximal disease-modifying effect for both amyloid pathology and cholinergic dysfunction.

Although mutations and variations of several genes have been identified to be involved in Alzheimer's disease (AD), the efforts towards understanding the pathogenic mechanisms of the disease still have a long journey to go. One such effort is to identify the signal transduction deficits, for which previous studies have suggested that the central problems appear to be at the interface between G proteins and their coupled receptors. G protein-coupled receptor kinases (GRKs) are a small family of serine/threonine protein kinases primarily acting at the “receptor-G protein interface””. Recent studies have indicated the possible involvement of GRK, primarily GRK2 and GRK5, dysfunction in the pathogenesis of AD. It seems that mild, soluble, β-amyloid accumulation can lead to a reduced membrane (functional) and an elevated cytosolic GRK2/5. The increased cytosolic GRK2 appears to be colocalized with damaged mitochondria and neurofibrillary tangles. Moreover, the total levels of GRK2, not only in the brain, but also in peripheral blood samples, are increased in a manner inversely correlated with the patient's cognitive levels. The deficiency of GRK5, on the other hand, impairs presynaptic M2 autoreceptor desensitization, which leads to a reduced acetylcholine release, axonal/synaptic degenerative changes, and associated amnestic, mild cognitive impairment. It also promotes an evil cycle to further increase Beta-amyloid accumulation and exaggerates brain inflammation, possibly even the basal forebrain cholinergic degeneration. Therefore, continuous efforts in this direction are necessary before translating the knowledge to any therapeutic strategies.

Recent studies suggested that G protein-coupled receptor kinase 5 (GRK5) deficiency plays a significant role in early Alzheimer's disease (AD) pathogenesis, and that the GRK5 knockout (GRK5KO) mouse displays an early Alzheimer-like cognitive deficit associated with increased hippocampal axonal defects and synaptic degenerative changes. Gender is known to play a role in AD, with females showing more extensive pathologic changes in brain compared to males. Although GRK5 deficiency is linked to AD, it is unknown whether the pathologic changes solely driven by the GRK5 deficiency are gender-dependent. To determine this, extent of the pathologic changes in aged GRK5KO mice was compared between genders. We find that female GRK5KO mice had a 2.5-fold increase in hippocampal swollen axonal clusters compared to male GRK5KO mice. Moreover, hippocampal levels of several synaptic proteins, including synaptophysin, were significantly lower in the female than the male. In addition, although increased Luteinizing hormone (LH) activity is believed to play a significant role in the gender phenomenon in AD, we found that desensitization of LH receptor is not affected by the GRK5 deficiency. Therefore, the worsened pathologic changes in the female mice cannot be attributed to an impaired LH receptor desensitization. Taken together, this study demonstrates a synergistic interaction between GRK5 deficiency and gender in promoting early AD-like pathologic changes in the female GRK5KO mice, although the underlying molecular mechanisms remain to be elucidated.

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.

Background Deficiency of membrane G-protein coupled receptor (GPCR) kinase-5 (GRK5) recently has been linked to early AD pathogenesis, and has been suggested to contribute to augmented microglial activation in vitro by sensitizing relevant GPCRs. However, GRK5 deficient mice did not show any signs of microgliosis, except for their moderate increase in axonal defects and synaptic degenerative changes during aging. We have speculated that one possible reason for the absence of microgliosis in these animals might be due to lack of an active inflammatory process involving activated GPCR signaling, since GRKs only act on activated GPCRs. The objective of this study was to determine whether the microgliosis is exaggerated in TgAPPsw (Tg2576) mice also deficient in GRK5, in which fibrillar β-amyloid (Aβ) and an active inflammatory process involving activated GPCR signaling are present. Methods Both quantitative and qualitative immunochemistry methods were used to evaluate the microgliosis and astrogliosis in these animals. Results We found that inactivation of one copy of the GRK5 gene in the TgAPPsw mice resulted in approximately doubled extent of microgliosis, along with significantly exaggerated astrogliosis, in both hippocampus and cortex of the aged animals. Consistent with previous observations, the activated microglia were located primarily near or surrounding the fibrillar Aβ deposits. Conclusion The results demonstrate that GRK5 deficiency in vivo significantly exaggerates microgliosis and astrogliosis in the presence of an inflammatory initiator, such as the excess fibrillar Aβ and the subsequent active inflammatory reactions in the TgAPPsw mice.