Investigating the role of peripheral serotonergic, purinergic and nitric oxide/cGMP pathways in the normal and pathological erectile process and the effect of Vardenafil on diabetic nephropathy

Serotonin (5-HT, 5-hydroxytrptamine), purinergic and nitric oxide (NO)/ cyclic guanosine monophosphate (cGMP) pathways have been implicated in human penile erection and erectile dysfunction (ED) and the later also involved in the pathogenesis of diabetic nephropathy (DN). The role and characterisation of the peripheral serotonergic and purinergic pathway in corpus cavernosal smooth muscle (CCSM) function was investigated. Electrical field stimulation of human and rabbit CCSM strips demonstrated the presence of neuronally-derived 5-HT acting on 5-HT(2A) receptors. Organ bath studies also showed that 5-HT caused CCSM contraction via 5-HT(1A), (1B), (2A) and (4) receptor subtypes, which was inhibited by doxazosin (an α-1 receptor antagonist known to have 5-HT inhibitory action). Immunohistochemistry and Western Blot analysis confirmed the presence of these 5-HT receptor subtypes in human CCSM. The effect of 5-HT and sodium nitroprusside (SNP; NO donor) on CCSM function from normal, diabetic and partial bladder outlet obstructed (PBOO) rabbit models were also assessed as these pathological models are associated with ED. There was no difference in 5-HT-induced CCSM contractions between the control and pathological models. In contrast, SNP-mediated relaxations were impaired in diabetic and PBOO rabbits. Vardenafil (phosphodiesterase type-5 inhibitor) improved the impaired CCSM relaxation in both models. Immunohistochemistry also identified the presence of purinergic P2Y6 receptors in human CCSM, which when activated induced tissue relaxation. Finally, the effect oral vardenafil treatment has on established renal impairment in diabetic rabbits was investigated. Vardenafil increased cGMP accumulation and improved renal function in DN. These findings suggests that targeting the peripheral serotonergic and purinergic pathways may be useful therapeutic options in treating ED. In addition, the use of vardenafil may be beneficial in the management of DN

A primary rodent triculture model to investigate the role of glia-neuron crosstalk in regulation of neuronal activity

Neuroinflammation and hyperexcitability have been implicated in the pathogenesis of neurodegenerative disease, and new models are required to investigate the cellular crosstalk involved in these processes. We developed an approach to generate a quantitative and reproducible triculture system that is suitable for pharmacological studies. While primary rat cells were previously grown in a coculture medium formulated to support only neurons and astrocytes, we now optimised a protocol to generate tricultures containing neurons, astrocytes and microglia by culturing in a medium designed to support all three cell types and adding exogenous microglia to cocultures. Immunocytochemistry was used to confirm the intended cell types were present. The percentage of ramified microglia in the tricultures decreases as the number of microglia present increases. Multi-electrode array recordings indicate that microglia in the triculture model suppress neuronal activity in a dose-dependent manner. Neurons in both cocultures and tricultures are responsive to the potassium channel blocker 4-aminopyridine, suggesting that neurons remained viable and functional in the triculture model. Furthermore, suppressed neuronal activity in tricultures correlates with decreased densities of dendritic spines and of the postsynaptic protein Homer1 along dendrites, indicative of a direct or indirect effect of microglia on synapse function. We thus present a functional triculture model, which, due to its more complete cellular composition, is a more relevant model than standard cocultures. The model can be used to probe glia-neuron interactions and subsequently aid the development of assays for drug discovery, using neuronal excitability as a functional endpoint

Disruption of endoplasmic reticulum-mitochondria tethering proteins in post-mortem Alzheimer's disease brain

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions, many of which are perturbed in Alzheimer's disease. Moreover, damage to ER-mitochondria signaling is seen in cell and transgenic models of Alzheimer's disease. However, as yet there is little evidence that ER-mitochondria signaling is altered in human Alzheimer's disease brains. ER-mitochondria signaling is mediated by interactions between the integral ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 which act to recruit and “tether” regions of ER to the mitochondrial surface. The VAPB-PTPIP51 tethers are now known to regulate a number of ER-mitochondria signaling functions including delivery of Ca2+from ER stores to mitochondria, mitochondrial ATP production, autophagy and synaptic activity. Here we investigate the VAPB-PTPIP51 tethers in post-mortem control and Alzheimer's disease brains. Quantification of ER-mitochondria signaling proteins by immunoblotting revealed loss of VAPB and PTPIP51 in cortex but not cerebellum at end-stage Alzheimer's disease. Proximity ligation assays were used to quantify the VAPB-PTPIP51 interaction in temporal cortex pyramidal neurons and cerebellar Purkinje cell neurons in control, Braak stage III-IV (early/mid-dementia) and Braak stage VI (severe dementia) cases. Pyramidal neurons degenerate in Alzheimer's disease whereas Purkinje cells are less affected. These studies revealed that the VAPB-PTPIP51 tethers are disrupted in Braak stage III-IV pyramidal but not Purkinje cell neurons. Thus, we identify a new pathogenic event in post-mortem Alzheimer's disease brains. The implications of our findings for Alzheimer's disease mechanisms are discussed

LMTK2 binds to kinesin light chains to mediate anterograde axonal transport of cdk5/p35 and LMTK2 levels are reduced in Alzheimer's disease brains

Cyclin dependent kinase-5 (cdk5)/p35 is a neuronal kinase that regulates key axonal and synaptic functions but the mechanisms by which it is transported to these locations are unknown. Lemur tyrosine kinase-2 (LMTK2) is a binding partner for p35 and here we show that LMTK2 also interacts with kinesin-1 light chains (KLC1/2). Binding to KLC1/2 involves a C-terminal tryptophan/aspartate (WD) motif in LMTK2 and the tetratricopeptide repeat (TPR) domains in KLC1/2, and this interaction facilitates axonal transport of LMTK2. Thus, siRNA loss of KLC1 or mutation of the WD motif disrupts axonal transport of LMTK2. We also show that LMTK2 facilitates the formation of a complex containing KLC1 and p35 and that siRNA loss of LMTK2 disrupts axonal transport of both p35 and cdk5. Finally, we show that LMTK2 levels are reduced in Alzheimer’s disease brains. Damage to axonal transport and altered cdk5/p35 are pathogenic features of Alzheimer’s disease. Thus, LMTK2 binds to KLC1 to direct axonal transport of p35 and its loss may contribute to Alzheimer’s disease

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by “tethering proteins” that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson’s disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson’s disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson’s disease and FTD/ALS

ALS/FTD-associated FUS activates GSK-3 to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria associations

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca2+ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca2+ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD

Brain energy rescue:an emerging therapeutic concept for neurodegenerative disorders of ageing

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner — a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes

Triple-negative breast cancer in Hong Kong Chinese patients

Background: There is increasing data showing that breast cancer is a heterogeneous disease which should be assessed separately in different populations, as it differs substantially between Chinese and Caucasian women. Triple-negative breast tumours which are negative for ER, PR and HER-2 neu receptors are associated with younger age at presentation, tumour of higher grade with larger size and a poorer prognosis. There is recent suggestion that the prognostic outlook of Chinese triple-negative breast cancers might be somewhat different from those in the Western population, but few studies have attempted to understand the role of ethnic factor in the triple-negative entity. Methods: We conducted a preliminary retrospective comparison of 170 Hong Kong Chinese primary breast cancer patients seen as new cases during January 2004 and December 2004 in a teaching hospital. Clinico-pathological features of triple-negative tumours were compared to their non-triple-negative counterpart. Results: Triple negative breast cancer accounted for 12.4% of all breast cancer patients seen in the year of 2004 (n = 21). It is associated with more cancers with grade 3 tumour (68.4% vs 36.8%; p = 0.02) but there was no statistically difference between the age of presentation, tumour size, extensive intraductal component, lymph node status and rate of local relapse or metastasis after adjuvant therapy. Subset analysis further revealed that when triple negative breast cancer patients (n = 21) were compared to the HER-2 positive patients (n = 40) in the studied population, HER-2 positive patients were still associated with higher proportion of node positive disease (57.5% vs 30.0%; p = 0.04). Disease free survival and overall survival were not studied due to limited follow-up time. Conclusions: Our preliminary findings suggested that Hong Kong Chinese triple-negative breast cancers are associated with a more favourable outlook and might behave differently when compared to their Western counterpart. Further large-scale study of the ethnic factor with long-term follow-up is warranted