Recently, a panel of experts gathered by the National Institute of Aging and the Alzheimer's Association published new biological guidelines for the definition of Alzheimer's disease (AD) rather than the symptomatic approach typically used in the clinic. Such a change was judged necessary, considering that no FDA-approved AD treatments currently address the main hallmarks of the disease. With the recent clinical trial shortcomings of AD immunotherapy, γ- and β-secretase inhibitors, among other interventions, we and colleagues agree that a multifactorial approach is needed to address the polygenicity of AD. We used an epigenetic strategy where a single drug would simultaneously affect the expression of well-defined AD-related targets. AD-related genes and proteins were analyzed using NanoString technology, RT-qPCR methods, Western blots and ELISAs (N = 3 to 6). Behavioral effects on the 3xTg AD mouse model were assessed using the open field, the Y-maze, the Barnes Maze and the novel object recognition tests (N=10). Unpaired Student's T-test was used whenever only two means were being compared. One-way ANOVA or repeated measures two-way ANOVA with appropriate post hoc analyses were used for multiple comparisons. Pan and selective HDAC inhibition both reduce Aβ(1-42) accumulation (p<0.05), decrease tau phosphorylation (p<0.01), and increase the expression of several high-priority AD-related protective genes. For example, compound M344 normalizes late-onset risk factor AD genes such as APOEe4 (p<0.0001) and BIN1 (p<0.0001). M344 also results in prevention of cognitive impairment in the 3xTg AD mice. M344 shows low toxicity, and rapidly clears out of brain and plasma. Similar positive results are also obtained with a more selective HDAC inhibitor compound. Our data suggest that brief daily brain exposure of a small epigenetic molecule can target the non-amyloidogenic pathway, increase neuroprotective genes, show low toxicity and increase memory in an AD model. This work supports a shift to a multitargeted approach to the treatment of AD.

None of the FDA-approved Alzheimer’s disease (AD) treatments address the main hallmarks of the disease. With the recent clinical trial shortcomings of AD immunotherapy as well as γ- and β-secretase inhibitors among others, we and colleagues agree that a multifactorial approach is needed to address the polygenicity of AD. We used an epigenetic strategy where a single drug would simultaneously affect the expression of well-defined AD-related targets.

Significance Hundreds of failed clinical trials with Alzheimer’s disease (AD) patients over the last fifteen years demonstrate that the one-target–one-disease approach is not effective in AD. In silico, structure-based, multitarget drug design approaches to treat multifactorial diseases have not been successful in the context of AD either. Here, we show that M344, an inhibitor of class I and IIB histone deacetylases, affects multiple AD-related genes, including those related to both early- and late-onset AD. We also show that M344 improves memory in the 3xTg AD mouse model. This work endorses a shift to a multitargeted approach to the treatment of AD, supporting the therapeutic potential of a single small molecule with an epigenetic mechanism of action.

The amygdala-dependent molecular mechanisms driving the onset and persistence of posttraumatic stress disorder (PTSD) are poorly understood. Recent observational studies have suggested that opioid analgesia in the aftermath of trauma may decrease the development of PTSD. Using a mouse model of dysregulated fear, we found altered expression within the amygdala of the Oprl1 gene (opioid receptor-like 1), which encodes the amygdala nociceptin (NOP)/orphanin FQ receptor (NOP-R). Systemic and central amygdala infusion of SR-8993, a new highly selective NOP-R agonist, impaired fear memory consolidation. In humans, a single-nucleotide polymorphism (SNP) within OPRL1 is associated with a self-reported history of childhood trauma and PTSD symptoms (n = 1847) after a traumatic event. This SNP is also associated with physiological startle measures of fear discrimination and magnetic resonance imaging analysis of amygdala-insula functional connectivity. Together, these data suggest that Oprl1 is associated with amygdala function, fear processing, and PTSD symptoms. Further, our data suggest that activation of the Oprl1/NOP receptor may interfere with fear memory consolidation, with implications for prevention of PTSD after a traumatic event.

Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (for example, excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein-coupling partners. In the basolateral amygdala of fear-naive mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1–G protein coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.

Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M-1 subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60-80%) reductions in cortical [H-3]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [S-35]-GTPgS-G alpha(q/11) immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased G alpha(q/11)-[S-35]-GTPgS binding, with AC-42 producing responses that were similar to 50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate G alpha(q/11)-[S-35]-GTPgS binding was significantly decreased in the MRDS group (pEC(50) (M) = 5.69 +/- 0.16) compared with the control group (6.17 +/- 0.10) and the non-MRDS group (6.05 +/- 0.07). The levels of G alpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated G alpha(q/11)-[35 S]-GTPgS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([S-35]-GTPgS binding to G alpha(q/11)) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls. Furthermore, in a subgroup of patients with schizophrenia displaying markedly decreased PZP binding (MRDS) we have shown that although agonist potency may decrease, the efficacy of CHRM1-G alpha(q/11) coupling increases, suggesting an adaptative change in receptor-G protein coupling efficiency in this endophenotype of patients with schizophrenia. Neuropsychopharmacology (2009) 34, 2156-2166; doi:10.1038/npp.2009.41; published online 29 April 2009

Using a selective Galpha(q/11) protein antibody capture guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding approach, it has been possible to perform a quantitative pharmacological examination of the functional activity of the M(1) muscarinic acetylcholine receptor (mAChR) in membranes prepared from human postmortem cerebral cortex. Oxotremorine-M caused a > or = 2-fold increase in [35S]GTPgammaS-Galpha(q/11) binding with a pEC(50) of 6.06 +/- 0.16 in Brodmann's areas 23 and 25 that was almost completely inhibited by preincubation of membranes with the M(1) mAChR subtype-selective antagonist muscarinic toxin-7. In addition, the orthosteric and allosteric agonists, xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine] and AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), increased [35S]-GTPgammaS-Galpha(q/11) binding, but with reduced intrinsic activities, inducing maximal responses that were 42 +/- 1 and 44 +/- 2% of the oxotremorine-M-induced response, respectively. These data indicate that the M(1) receptor is the predominant mAChR subtype coupling to the Galpha(q/11) G protein in these brain regions and that it is possible to quantify the potency and intrinsic activity of full and partial M(1) mAChR receptor agonists in postmortem human brain using a selective Galpha(q/11) protein antibody capture [35S]GTPgammaS binding assay.

Increased platelet reactivity has been implicated in the vascular complications of myeloproliferative diseases and diabetes mellitus. The mechanisms of platelet hyperresponsiveness have not been fully explained. Expression of CD36 or fatty acid translocase (FAT) and its role in arachidonic acid (AA) uptake by platelets were examined in subjects with myeloproliferative disorders(MPD), those with non-insulin-dependent diabetes mellitus (NIDDM), and in normal, healthy, age-matched controls. Surface expression of CD36 on platelet membranes was increased in MPD (10.94 ± 0.76 pmol/mg protein) compared with normal controls (6.94 ± 0.48 pmol/mg protein), p < 0.001. Total platelet content of CD36 was also significantly higher (32.1 ± 0.61 pmol/mg protein, p < 0.01) compared with those in sex and age matched normal controls (25.7 ± 1.09 pmol/mg protein). In contrast, platelet surface expression of CD36 in NIDDM (6.5 ± 0.56 pmol/mg protein) was not significantly different from those of normal controls despite higher total content of CD36 (32.8 ± 1.2, pmol/mg protein, p < 0 .01). Intact MPD platelets bound significantly more arachidonic acid (AA) (1.53 ± 0.16 nmol/mg protein, p < 0.05), compared with controls (1.12 ± 0.07 nmol/mg protein) or NIDDM subjects (1.16 ± 0.16 nmol/mg protein). The capacity of MPD platelet membranes to bind 14C-AA was also increased (1.72 ± 0.25 nmol/ protein, p < 0.05) compared with that of controls (1.62 ± 0.05 nmol/protein) and of NIDDM (1.22 ± 0.08 nmol/protein). This is consistent with higher surface expression of CD36 in MPD platelets. Membrane fatty acid analysis indicated that the % of AA in platelet phospholipids was significantly lower in MPD (3.15 ± 0.81%) compared with the controls (5.62 ± 1.7%, p < 0.05. The AA content of diabetic platelets (4.82 ± 1.1%) was not significantly different from normal controls. In summary, both total and surface expression of CD36 are increased in MPD, consistent with an enhanced capacity for uptake of AA by platelets. Increased expression of CD36 in platelets may play a role in the vaso-occlusive manifestations of MPD.

1Rowett Research Institute Aberdeen, University of Aberdeen, Scotland 2Dept. of Medicine & Therapeutics, University of Aberdeen, Scotland