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Prevention of Alzheimer’s disease (AD) is a high priority mission while searching for a disease modifying therapy for AD, a devastating major public health crisis. Clinical observations have identified a prodromal stage of AD for which the patients have mild cognitive impairment (MCI) though do not yet meet AD diagnostic criteria. As an identifiable transitional stage before the onset of AD, MCI should become the high priority target for AD prevention, assuming successful prevention of MCI and/or its conversion to AD also prevents the subsequent AD. By pulling this string, one demonstrated cause of amnestic MCI appears to be the deficiency of G protein-coupled receptor-5 (GRK5). The most compelling evidence is that GRK5 knockout (GRK5KO) mice naturally develop into aMCI during aging. Moreover, GRK5 deficiency was reported to occur during prodromal stage of AD in CRND8 transgenic mice. When a GRK5KO mouse was crossbred with Tg2576 Swedish amyloid precursor protein transgenic mouse, the resulted double transgenic GAP mice displayed exaggerated behavioral and pathological changes across the spectrum of AD pathogenesis. Therefore, the GRK5 deficiency possesses unique features and advantage to serve as a prophylactic therapeutic target for MCI due to AD.

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Donepezil (DNPZ) is one of the few FDA-approved widely used medication in the clinical care of Alzheimer's disease (AD) patients. To investigate the effect of geometry and to find the significance of an enol form if any in DNPZ on acetylcholinesterase (AChE) inhibition, we changed the tetrahedral geometry of DNPZ to planar trigonal pyramidal geometry by replacing the α-carbon atom next to ketone functionality with a nitrogen atom. To mimic 1-indanone in DNPZ, we selected 1-isoindolinone framework to synthesize 25 new DNPZ derivatives and characterized using 1H NMR, 13C NMR and ESI-MS spectroscopy methods. Drug likeliness profile for each compound was predicted using Molinspiration online software following Lipinski's rule. Commercially available assay kits were used to measure AChE and butyrylcholinesterase (BuChE) inhibitory effects. NIH/3T3 mouse embryonic fibroblast cell line was used to measure cytotoxic and proliferation effects using LDH and MTT assay, respectively. Compound #20 was selected for comparative computational docking, modelling and physicochemical studies. Our results show that DNPZ with tetrahedral geometry has 3-fold higher AChE inhibition as compared to compound #20 with planar trigonal pyramidal geometry. Our approach may be useful as a novel indirect method to study the significance of the enol form in DNPZ (or similar compounds), since constant interconversion between the keto and enol forms does not permit a direct determination of the effect of the enol form of DNPZ in vivo. Overall, we conclude that the tetrahedral is a better fit and any change in geometry significantly drives down the cholinesterase inhibitory effect of DNPZ.

Background: Ovarian cancer (OC), the most lethal gynecologic malignancy, is highly resistant to current treatment strategies. High-grade serous epithelial ovarian cancer (HGSOC) cells with increased somatic mutations and genomic instability and the resulting heterogeneous mutant phenotypes are highly resistant to therapy. Plant-derived natural products, including Amla (Emblica officinalis) extract (AE), have demonstrated potent anti-neoplastic properties. Recently we demonstrated that AE inhibits cell growth and the expression of angiogenic factors in OVCAR3 and SKOV3 OC cells in vitro as well as in xenografts in vivo. The goal of this study was to determine the anti-proliferative, anti-angiogenic and anti-metastatic effects of AE on carboplatinum- and taxol-resistant HGSOC cells carrying p53, BRCA1/2 mutations. Methods: Anti-proliferative and anti-metastatic effects of AE on recently characterized carboplatinum- and taxol-resistant HGSOC cells (TOV3041G, OV866(2), OV4453 and, OV4485) was determined using the MTT, migration, invasion and spheroid assays in vitro. To understand the mechanism of AE-induced changes in angiogenesis-related hypoxia-inducible factor 1α (HIF-1α) and insulin growth factor receptor 1 (IGF1R), and EMT-associated SNAIL1 and E-cadherin proteins were studied using immunostaining and Western blotting. In vivo effects of AE were determined using mouse xenograft tumor model of OC developed by subcutaneous injection of OV4485 cells that carry mutant p53 and BRCA1, most aggressive and resistant among HGSOC cell lines used in this study. Tumor growth was measured using morphometry. Immunostaining and Western blotting were used to determine changes in Ki67 (proliferation marker), CD31 (angiogenesis marker) as well as changes in HIF-1α, IGF1R, SNAIL1 and E-cadherin proteins. Results: AE significantly attenuated migration and invasiveness properties of all tested HGSOC cell phenotypes (P≤0.001), significantly reduced the expression of HIF-1α, IGF1R, and SNAIL1 and increased the expression of E-cadherin in all tested HGSOC cell lines (P=60%) that derived from OV4855 cells and decreased the expression of endothelial cell antigen-CD31, HIF-1α, IGF1R and SNAIL1 and increased the expression of E-cadherin in tumor tissues. Conclusions: AE sensitizes platinum- and taxol-resistant heterogenous HGSOC cells carrying mutations in p53, BRCA1/2 genes, and attenuates their malignant characteristics through targeting key signaling mechanisms of angiogenesis and metastasis. AE is a potential adjunct therapeutic agent for treating resistant, mutant, heterogenous OC.

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.

Chalcone [(E)-1,3-diphenyl-2-propene-1-one], a small molecule with α, β unsaturated carbonyl group is a precursor or component of many natural flavonoids and isoflavonoids. It is one of the privileged structures in medicinal chemistry. It possesses a wide range of biological activities encouraging many medicinal chemists to study this scaffold for its usefulness to oncology, infectious diseases, virology and neurodegenerative diseases including Alzheimer’s disease (AD). Small molecular size, convenient and cost-effective synthesis, and flexibility for modifications to modulate lipophilicity suitable for blood brain barrier (BBB) permeability make chalcones a preferred candidate for their therapeutic and diagnostic potential in AD. This review summarizes and highlights the importance of chalcone and its analogs as single target small therapeutic agents, multi-target directed ligands (MTDLs) as well as molecular imaging agents for AD. The information summarized here will guide many medicinal chemist and researchers involved in drug discovery to consider chalcone as a potential scaffold for the development of anti-AD agents including theranostics.

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.

Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ~54 kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe(2)SO(4) also increased the ~54 kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.

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.

Neurological diseases such as stroke, encephalitis, neurologic tumors, and neurodegenerative diseases are far from alien to us. These diseases are refractory, harmful, and sometimes devastating. The pathogenesis of some nervous system diseases remains elusive, especially the neurodegenerative diseases such as AD and PD. Many therapeutic strategies have been developed to potentially intervene in these progressive neurodegenerative events and minimize damage to the CNS. Despite the promising results that these researches have shown in in vivo and in vitro studies, challenges still remain, particularly when it comes to clinical translation. The causes are various, which may include biologic and physiologic differences between the preclinical models and the human condition and discrepancies in dosing and the timing of drug therapy. The last few years have witnessed an explosion in the use of genes and cells as biomedicines. Gene and cell therapies are researches with the goals of repairing the direct cause of genetic diseases in the DNA or cellular population, respectively. In many diseases, gene and cell therapies are combined in the development of promising therapies, which have helped provide reagents, concepts, and techniques that are elucidating the finer points of gene regulation, stem cell lineage, regenerative capacity, and remodeling. Additionally, increasing studies of novel drug delivery systems bring more considerable future on gene and cell therapies. This special issue mainly focuses on the latest ideas, developments, and applications in the field of gene and cell therapies for neurological disorders, most notably development of highly efficient gene expression and delivery system including viral and nonviral gene vectors for tissue repair in the nervous system; exploration and evaluation of the efficiency of new ideal routes of administration of gene and cells into brain; evaluation of appropriate biomaterials in terms of biocompatibility and biodegradability, biomechanical competency, and functionalization with instructive cues to guide neuroregeneration; tissue engineering that integrates cell therapies and scaffolding technologies to replace damaged neurons and to reestablish the coaxial connection of neuronal circuits for functional recovery; assessment of the safety and toxic side effects with gene and cell therapies; latest technologies of noninvasive in vivo imaging to track the transplanted gene or cells; new insights into the pathogenesis of neurological disorders by using cellular or animal models. In the accepted papers, researches of pathogenesis of some diseases, such as Aβ-induced autophagy in AD and ischemia-reperfusion injury induced apoptosis in stroke, were discussed. Studying specific aptamer for GP73 through systematic evolution of ligands by exponential enrichment and identifying its application in detecting hepatocellular carcinoma might provide a novel molecular probe for cell therapy. In vivo targeted MR imaging of endogenous neural stem cells (NSCs) in adult mouse brain by using certain nanoparticles offered a promising molecular probe in NSCs research. Moreover, the role of Nogo-A in PD and NTRK1 in NSCs differentiation was preliminarily explored. These findings above provided new insights into the pathogenesis of certain diseases and also represented part of the latest ideas, developments, and applications in the field of gene and cell therapies for neurological disorders. Jun Liu William Z. Suo Xing-Mei Zhang Chen Zhang Gongxiong Wu

In vivo electroporation is one of the most efficient methods for introducing the nucleic acids into the target tissues, and thus plays a pivotal role in gene therapeutic studies. In vivo electroporation in rodent brains is often involved in injection of nucleic acids into the brain ventricle or specific area and then applying appropriate electrical field to the correct area. Better understanding of the progress of electroporation in rodent brain may further facilitate gene therapeutic studies on some brain disorders. For this purpose, we briefly summarized the advantages, the procedures and recent progress of transferring nucleic acids into the rodent brain using in vivo electroporation.

Neurotoxic homocysteine (Hcy) is thought to be an independent risk factor for neurodegenerative diseases, including Alzheimer's disease. This study is to determine whether HT22 cells, a murine hippocampal neuronal model, can be used as an in vitro model, besides the primary neuronal cultures, to investigate the effects of Hcy.MTS assay and Hoechst 33342/propidium iodide discrimination were used to assess the cell viability and cell death on undifferentiated and differentiated HT22 cells. Semi-quantitative reverse transcription polymerase chain reaction and western blot were used to determine the expression of N-methyl D-aspartate (NMDA) receptor.We found that undifferentiated and differentiated HT22 cells responded to Hcy toxicity differentially, with the undifferentiated cells resistant while the differentiated cells sensitive. The underlying mechanism appeared to be the differential expression levels of NMDA glutamate receptor between the undifferentiated and differentiated cells. Similar to what have been observed in primary neuronal cultures, the Hcy toxicity in the differentiated HT22 cells was largely attenuated by NMDA receptor antagonists, MK-801 and memantine.These results suggest for the first time that the differentiation of HT22 cells could induce the expression of NMDA receptors, which lead to Hcy mediate concentration-dependent apoptosis-necrotic continuum of HT22 cell death. The differentiation status of the HT22 cells is important for modeling neurons in vitro, with the differentiated HT22 neurons resembling more characteristics of primary hippocampal neurons while the undifferentiated HT22 cells being proliferating neuronal precursor cells. The differentiated HT22 neurons can be used as a platform to study Hcy toxicity.

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.

Aims Hippocampal cholinergic hypofunction is known to be involved in the cognitive deficits of Alzheimer's disease, but the detailed mechanisms remain to be elucidated. In order to establish an in vitro hippocampal cholinergic neuronal model for the relevant mechanistic studies, we have characterized a widely used hippocampal neuronal cell line, HT22, a sub-line derived from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture. Main methods Western blot and immunocytochemistry were used to examine expression of cholinergic markers in HT22 cells. High potassium-evoked [3H]ACh release was used to evaluate the cholinergic functional properties of the cells. Key findings We found that HT22 cells express essential cholinergic markers, such as the high affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter, and muscarinic acetylcholine receptors. Exposure of HT22 cells to high potassium evoked [3H]ACh release in a dose-dependent manner. In addition, the [3H]ACh release was significantly potentiated when presynaptic autoreceptors were blocked. Significance Our results suggest that HT22 cells possess functional cholinergic properties, and can be used for an in vitro model for defining the mechanisms in cognitive deficits of Alzheimer's disease.

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.