Ligands of the translocator protein (TSPO) elicit pleiotropic neuroprotective effects that represent emerging treatment strategies for several neurodegenerative conditions. To investigate the potential of TSPO as a therapeutic target for Alzheimer's disease (AD), the current study assessed the effects of the TSPO ligand Ro5-4864 on the development of neuropathology in 3xTgAD mice. The effects of the TSPO ligand on neurosteroidogenesis and AD-related neuropathology including β-amyloid accumulation, gliosis and behavioral impairment were examined under both early intervention (7 month-old young-adult male mice with low pathology) and treatment (24 month-old, aged male mice with advanced neuropathology) conditions. Ro5-4864 treatment not only effectively attenuated development of neuropathology and behavioral impairment in young adult mice, but also reversed these indices in aged 3xTgAD mice. Reduced levels of soluble β-amyloid were also observed by the combination of TSPO ligands Ro5-4864 and PK11195 in non-transgenic mice. These findings suggest TSPO is a promising target for the development of pleiotropic treatment strategies for the management of AD.

Obesity, metabolic syndrome, and type 2 diabetes (T2D) are related disorders with widespread deleterious effects throughout the body. One important target of damage is the brain. Persons with metabolic disorders are at significantly increased risk for cognitive decline and the development of vascular dementia and Alzheimer’s disease. Our review of available evidence from epidemiological, clinical, and basic research suggests that neural dysfunction from T2D-related disease results from several underlying mechanisms, including metabolic, inflammatory, vascular and oxidative changes. The relationships between T2D and neural dysfunction are regulated by several modifiers. We emphasize two such modifiers, the genetic risk factor apolipoprotein E and an age-related endocrine change, low testosterone. Both factors are independent risk factors for Alzheimer’s disease that may also cooperatively regulate pathologic interactions between T2D and dementia. Continued elucidation of the links between metabolic disorders and neural dysfunction promises to foster the development of effective therapeutic strategies.

Sustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II–III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I–III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.

The accumulation of β-amyloid protein (Aβ) is a key risk factor in the development of Alzheimer's disease. The ovarian sex steroid hormones 17β-estradiol (E(2)) and progesterone (P(4)) have been shown to regulate Aβ accumulation, although the underlying mechanism(s) remain to be fully elucidated. In this study, we investigate the effects of E(2) and P(4) treatment on the expression levels of Aβ clearance factors including insulin-degrading enzyme, neprilysin, endothelin-converting enzyme 1 and 2, angiotensin-converting enzyme, and transthyretin, both in primary neuron cultures and female rat brains. Our results show that E(2) and P(4) affect the expression levels of several Aβ clearance factors in dose- and time-dependent manners. Most notably, expression of insulin-degrading enzyme is significantly increased by both hormones in cultured neurons and in vivo and is inversely associated with the soluble Aβ levels in vivo. These findings further define sex steroid hormone actions involved in regulation of Aβ, a relationship potentially important to therapeutic approaches aimed at reducing risk of Alzheimer's disease.

The pathogenetic mechanisms underlying neuronal death and dysfunction in Alzheimer’s disease (AD) remain unclear. However, chronic neuroinflammation has been implicated in stimulating or exacerbating neuronal damage. The tumor necrosis factor (TNF) superfamily of cytokines are involved in many systemic chronic inflammatory and degenerative conditions and are amongst the key mediators of neuroinflammation. TNF binds to the TNFR1 and TNFR2 receptors to activate diverse cellular responses that can be either neuroprotective or neurodegenerative. In particular, TNF can induce programmed necrosis or necroptosis in an inflammatory environment. Although activation of necroptosis has recently been demonstrated in the AD brain, its significance in AD neuron loss and the role of TNF signaling is unclear. We demonstrate an increase in expression of multiple proteins in the TNF/TNF receptor-1-mediated necroptosis pathway in the AD post-mortem brain, as indicated by the phosphorylation of RIPK3 and MLKL, predominantly observed in the CA1 pyramidal neurons. The density of phosphoRIPK3 + and phosphoMLKL + neurons correlated inversely with total neuron density and showed significant sexual dimorphism within the AD cohort. In addition, apoptotic signaling was not significantly activated in the AD brain compared to the control brain. Exposure of human iPSC-derived glutamatergic neurons to TNF increased necroptotic cell death when apoptosis was inhibited, which was significantly reversed by small molecule inhibitors of RIPK1, RIPK3, and MLKL. In the post-mortem AD brain and in human iPSC neurons, in response to TNF, we show evidence of altered expression of proteins of the ESCRT III complex, which has been recently suggested as an antagonist of necroptosis and a possible mechanism by which cells can survive after necroptosis has been triggered. Taken together, our results suggest that neuronal loss in AD is due to TNF-mediated necroptosis rather than apoptosis, which is amenable to therapeutic intervention at several points in the signaling pathway.

BackgroundLow testosterone and obesity are independent risk factors for dysfunction of the nervous system including neurodegenerative disorders such as Alzheimer’s disease (AD). In this study, we investigate the independent and cooperative interactions of testosterone and diet-induced obesity on metabolic, inflammatory, and neural health indices in the central and peripheral nervous systems.MethodsMale C57B6/J mice were maintained on normal or high-fat diet under varying testosterone conditions for a four-month treatment period, after which metabolic indices were measured and RNA isolated from cerebral cortex and sciatic nerve. Cortices were used to generate mixed glial cultures, upon which embryonic cerebrocortical neurons were co-cultured for assessment of neuron survival and neurite outgrowth. Peripheral nerve damage was determined using paw-withdrawal assay, myelin sheath protein expression levels, and Na+,K+-ATPase activity levels.ResultsOur results demonstrate that detrimental effects on both metabolic (blood glucose, insulin sensitivity) and proinflammatory (cytokine expression) responses caused by diet-induced obesity are exacerbated by testosterone depletion. Mixed glial cultures generated from obese mice retain elevated cytokine expression, although low testosterone effects do not persist ex vivo. Primary neurons co-cultured with glial cultures generated from high-fat fed animals exhibit reduced survival and poorer neurite outgrowth. In addition, low testosterone and diet-induced obesity combine to increase inflammation and evidence of nerve damage in the peripheral nervous system.ConclusionsTestosterone and diet-induced obesity independently and cooperatively regulate neuroinflammation in central and peripheral nervous systems, which may contribute to observed impairments in neural health. Together, our findings suggest that low testosterone and obesity are interactive regulators of neuroinflammation that, in combination with adipose-derived inflammatory pathways and other factors, increase the risk of downstream disorders including type 2 diabetes and Alzheimer’s disease.

J. Neurochem. (2010) 115, 1277–1287. Abstract While both 17β‐estradiol (E2) and progesterone (P4) are neuroprotective in several experimental paradigms, P4 also counteracts E2 neuroprotective effects. We recently reported that a 4‐h treatment of cultured hippocampal slices with P4 following a prolonged (20 h) treatment with E2 eliminated estrogenic neuroprotection against NMDA toxicity and induction of brain‐derived neurotrophic factor (BDNF) expression. In the present study, we evaluated the effects of the same treatment on levels of estrogen receptors, ERα and ERβ, and BDNF using a similar paradigm. E2 treatment resulted in elevated ERβ mRNA and protein levels, did not modify ERα mRNA, but increased ERα protein levels, and increased BDNF mRNA levels. P4 reversed E2‐elicited increases in ERβ mRNA and protein levels, in ERα protein levels, and in BDNF mRNA levels. Experiments with an ERβ‐specific antagonist, PHTPP, and specific agonists of ERα and ERβ, propylpyrazoletriol and diarylpropionitrile, respectively, indicated that E2‐mediated neuroprotection against NMDA toxicity was, at least in part, mediated via ERβ receptor. In support of this conclusion, E2 did not protect against NMDA toxicity in cultured hippocampal slices from ERβ−/− mice. Thus, E2‐mediated neuroprotection against NMDA toxicity may be because of estrogenic induction of BDNF via its ERβ receptor, and P4‐mediated inhibition of E2 neuroprotective effects treatment to P4‐induced down‐regulation of ERβ and BDNF.

Recent findings indicate that progesterone can attenuate beneficial neural effects of oestrogen. Here, we investigate the hypothesis that progesterone can modulate oestrogen actions by regulating expression and activity of oestrogen receptors, ERα and ERβ. Our studies in cultured neurones demonstrate that progesterone decreases the expression of both ERα and ERβ and, as a consequence, also reduces both ER-dependent transcriptional activity and neuroprotection. These results identify a potential mechanism by which progesterone antagonises neural oestrogen actions, a finding that may have important implications for hormone therapy in postmenopausal women. Keywordsoestrogen; oestrogen receptors; hormone therapy; neuroprotection; progesterone The depletion of oestrogen and progesterone in postmenopausal women is associated with increased risk for several disorders in the cardiovascular, skeletal and nervous systems (1). For example, the Women's Health Initiative clinical trial showed that HT use was associated with reduced incidence of hip fractures but, unexpectedly, increased incidences of both stroke and dementia (2). The disparities between basic research studies that demonstrate neuroprotective effects of oestrogen and recent clinical findings that report adverse neural effects of HT indicate the need for a more complete understanding of oestrogen and progesterone interactions in brain and other tissues. To gain some mechanistic insight into this issue, we studied the effects of progesterone on oestrogen actions in cultured neurones.One important issue that is not well understood is how neural effects of oestrogen are affected by progestagens. Recent experimental evidence in rodent models shows that prolonged progesterone (P4) exposure often represses beneficial 17β-oestradiol (E 2 ) function in the brain (3-9). The mechanism(s) by which P4 inhibits E 2 action in the brain is unclear. Here, we investigate the possibility that P4 modulates E 2 action by regulating expression of oestrogen receptors (ERs). We demonstrate that P4 treatment reduces the expression of both ERα and ERβ in cultured neurones in a concentration-and timedependent manner. We also show that this decrease in ER expression leads to attenuation of E 2 activity in the neurones. We investigated P4 regulation of ER expression and E 2 neuroprotection using an established neurone culture paradigm. Experimental animal procedures were conducted in accordance with the University of Southern California guidelines based on National Institute of Health standards. Primary rat cerebrocortical cultures (∼95% neuronal) were generated from gestational day 16-17 rat pups using a previously described protocol (10) with some modifications. Cultures were seeded in multiwell plates at final densities of approximately 2.5 × 10 4 cells/cm 2 (cell viability and luciferase assays) or 8 × 10 5 cells/cm 2 (RNA isolation) and experiments were started 1-2 days after plating. In all experiments, both E 2 (Steraloids Inc.; Newport, RI) and P4 (Acros Organics USA; Mor...