M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer's disease genes and improving memory.

Alzheimer's disease (AD) comprises multifactorial ailments for which current therapeutic strategies remain insufficient to broadly address the underlying pathophysiology. Epigenetic gene regulation relies upon multifactorial processes that regulate multiple gene and protein pathways, including those involved in AD. We therefore took an epigenetic approach where a single drug would simultaneously affect the expression of a number of defined AD-related targets. We show that the small-molecule histone deacetylase inhibitor M344 reduces beta-amyloid (Aß), reduces tau Ser396 phosphorylation, and decreases both ß-secretase (BACE) and APOEε4 gene expression. M344 increases the expression of AD-relevant genes: BDNF, α-secretase (ADAM10), MINT2, FE65, REST, SIRT1, BIN1, and ABCA7, among others. M344 increases sAPPα and CTFα APP metabolite production, both cleavage products of ADAM10, concordant with increased ADAM10 gene expression. M344 also increases levels of immature APP, supporting an effect on APP trafficking, concurrent with the observed increase in MINT2 and FE65, both shown to increase immature APP in the early secretory pathway. Chronic i.p. treatment of the triple transgenic (APPsw/PS1M146V/TauP301L) mice with M344, at doses as low as 3 mg/kg, significantly prevented cognitive decline evaluated by Y-maze spontaneous alternation, novel object recognition, and Barnes maze spatial memory tests. M344 displays short brain exposure, indicating that brief pulses of daily drug treatment may be sufficient for long-term efficacy. Together, these data show that M344 normalizes several disparate pathogenic pathways related to AD. M344 therefore serves as an example of how a multitargeting compound could be used to address the polygenic nature of multifactorial diseases.

Amygdala-dependent fear is regulated by Oprl1 in mice and humans with PTSD.

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.

Altered M(1) muscarinic acetylcholine receptor (CHRM1)-Galpha(q/11) coupling in a schizophrenia endophenotype.

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 [3H]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [35S]-GTPgammaS-Galpha(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 Galpha(q/11)-[35S]-GTPgammaS binding, with AC-42 producing responses that were approximately 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 Galpha(q/11)-[35S]-GTPgammaS 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 Galpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Galpha(q/11)-[35S]-GTPgammaS 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 ([35S]-GTPgammaS binding to Galpha(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-Galpha(q/11) coupling increases, suggesting an adaptative change in receptor-G protein coupling efficiency in this endophenotype of patients with schizophrenia.

Pharmacological assessment of m1 muscarinic acetylcholine receptor-gq/11 protein coupling in membranes prepared from postmortem human brain tissue.

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.

Surface expression of fatty acid translocase (FAT/CD36) on platelets in myeloproliferative disorders and non-insulin dependent diabetes mellitus: effect on arachidonic acid uptake.

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/mg protein, p < 0.05) compared with that of controls (1.62 +/- 0.05 nmol/mg protein) and of NIDDM (1.22 +/- 0.08 nmol/mg 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.