Editorial: sigma receptors.

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Highly Selective Butyrylcholinesterase Inhibitors with Tunable Duration of Action by Chemical Modification of Transferable Carbamate Units Exhibit Pronounced Neuroprotective Effect in an Alzheimer’s Disease Mouse Model

In this study, the carbamate structure of pseudo-irreversible butyrylcholinesterase (BChE) inhibitors was optimized with regard to a longer binding to the enzyme. A set of compounds bearing different heterocycles (e.g., morpholine, tetrahydroisoquinoline, benzimidazole, piperidine) and alkylene spacers (2 to 10 methylene groups between carbamate and heterocycle) in the carbamate residue was synthesized and characterized in vitro for their binding affinity, binding kinetics, and carbamate hydrolysis. These novel BChE inhibitors are highly selective for hBChE over human acetycholinesterase (hAChE), yielding short-, medium-, and long-acting nanomolar hBChE inhibitors (with a half-life of the carbamoylated enzyme ranging from 1 to 28 h). The inhibitors show neuroprotective properties in a murine hippocampal cell line and a pharmacological mouse model of Alzheimer’s disease (AD), suggesting a significant benefit of BChE inhibition for a disease-modifying treatment of AD

In vivo and ex vivo analyses of amyloid toxicity in the Tc1 mouse model of Down syndrome

RATIONALE: The prevalence of Alzheimer's disease is increased in people with Down syndrome. The pathology appears much earlier than in the general population, suggesting a predisposition to develop Alzheimer's disease. Down syndrome results from trisomy of human chromosome 21, leading to overexpression of possible Alzheimer's disease candidate genes, such as amyloid precursor protein gene. To better understand how the Down syndrome context results in increased vulnerability to Alzheimer's disease, we analysed amyloid-β [25-35] peptide toxicity in the Tc1 mouse model of Down syndrome, in which ~75% of protein coding genes are functionally trisomic but, importantly, not amyloid precursor protein. RESULTS: Intracerebroventricular injection of oligomeric amyloid-β [25-35] peptide in three-month-old wildtype mice induced learning deficits, oxidative stress, synaptic marker alterations, activation of glycogen synthase kinase-3β, inhibition of protein kinase B (AKT), and apoptotic pathways as compared to scrambled peptide-treated wildtype mice. Scrambled peptide-treated Tc1 mice presented high levels of toxicity markers as compared to wildtype mice. Amyloid-β [25-35] peptide injection in Tc1 mice induced significant learning deficits and enhanced glycogen synthase kinase-3β activity in the cortex and expression of apoptotic markers in the hippocampus and cortex. Interestingly, several markers, including oxidative stress, synaptic markers, glycogen synthase kinase-3β activity in the hippocampus and AKT activity in the hippocampus and cortex, were unaffected by amyloid-β [25-35] peptide injection in Tc1 mice. CONCLUSIONS: Tc1 mice present several toxicity markers similar to those observed in amyloid-β [25-35] peptide-treated wildtype mice, suggesting that developmental modifications in these mice modify their response to amyloid peptide. However, amyloid toxicity led to severe memory deficits in this Down syndrome mouse model

Highly Selective Butyrylcholinesterase Inhibitors with Tunable Duration of Action by Chemical Modification of Transferable Carbamate Units Exhibit Pronounced Neuroprotective Effect in an Alzheimer’s Disease Mouse Model

In this study, the carbamate structure of pseudo-irreversible butyrylcholinesterase (BChE) inhibitors was optimized with regard to a longer binding to the enzyme. A set of compounds bearing different heterocycles (e.g., morpholine, tetrahydroisoquinoline, benzimidazole, piperidine) and alkylene spacers (2 to 10 methylene groups between carbamate and heterocycle) in the carbamate residue was synthesized and characterized in vitro for their binding affinity, binding kinetics, and carbamate hydrolysis. These novel BChE inhibitors are highly selective for hBChE over human acetycholinesterase (hAChE), yielding short-, medium-, and long-acting nanomolar hBChE inhibitors (with a half-life of the carbamoylated enzyme ranging from 1 to 28 h). The inhibitors show neuroprotective properties in a murine hippocampal cell line and a pharmacological mouse model of Alzheimer’s disease (AD), suggesting a significant benefit of BChE inhibition for a disease-modifying treatment of AD

Tau phosphorylation.

<p>Effects of oAβ_25–35 (10 µg/rat) icv injection on Tau phosphorylation in the frontal cortex, amygdala, hippocampus and hypothalamus, determined by western blot in untreated control rats and 6 weeks after Aβ_25–35 scrambled amyloid peptide (10 µg/rat; negative control) or oAβ_25–35 icv injection. The Tau hyperphosphorylation (AT8; 50 KDa) and the abnormal Tau phosphorylation (AT100; 50 KDa) variations were expressed in function of total Tau expression (50 KDa) and compared with non-injected rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control non-injected rats (control group: C) and +p<0.05 and ++p<0.01 vs. respective scrambled peptide-treated rats. The number of animals in groups is indicated within the columns.</p

Physiological and behavioral effects of oAβ_25–35.

<p><b>A.</b> Body weight variations determined 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; scrambled group) or oAβ_25–35 (10 µg/rat; Aβ_25–35 group). The results are expressed as means ± SEM (with n = 6 per group). *p<0.05 <i>vs</i>. control value and +p<0.05 vs. scrambled value). <b>B.</b> Variations in locomotor activity and body temperature determined 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; scrambled group; n  = 7) or oAβ_25–35 (10 µg/rat; oAβ_25–35 group; n = 7). Locomotor activity and body temperature were monitored using telemetric sensors. The thick black line indicates the nocturnal phase (7:00 PM to 7:00 AM). The results are means/hour obtained the 6^th week following the icv injection. <b>C.</b> Effects of oAβ_25–35 icv injection (10 µg/rat) on the ability of rats to perform a spatial short-term memory task (T-maze). Six weeks after icv injection, animals were allowed to explore the T-maze, with one short arm closed (B), for 10 min. After a 1 h time interval, the pattern of exploration of the whole maze was recorded for 2 min. The icv injection of the scrambled Aβ_25–35 peptide (10 µg/rat) served as negative control. The results are expressed as means ± SEM. **p<0.01 <i>vs.</i> control un-injected rats, +p<0.05 and ++p<0.01 <i>vs.</i> scrambled treated rats. The number of animals in each group is indicated within the columns. <b>D.</b> Effects of oAβ_25–35 icv injection (10 µg/rat) on rat behavior in a spatial long-term memory test (Water-maze). Six weeks after icv injection, animals were allowed to swim for 90 s to find the training platform and 60 s without the platform for retention. The icv injection of the scrambled Aβ_25–35 peptide (10 µg/rat) served as negative control. The results are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. control un-injected rats, +p<0.05 and ++p<0.01 <i>vs.</i> scrambled treated rats. <b>E.</b> The probe test was performed 4 h after the last training trial in a single 60 s-duration swimming without platform. The presence in the training quadrant was analyzed over the chance level (25%): # p<0.05 and ## p<0.01. The number of animals in each group is indicated within the columns. <b>F.</b> Variations in plasmatic corticosterone (CORT) levels determined in rats 6 weeks after icv injection of Aβ_25–35 scrambled peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). The values are means ± SEM. **p<0.01 <i>vs</i>. control un-injected rats (control group: C) and ++p<0.01 <i>vs</i>. scrambled treated rats. The number of animals in each group is indicated within the columns.</p

Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine

Macroautophagy/autophagy is an essential process for cellular survival and is implicated in many diseases. A critical step in autophagy is the transport of the transcription factor TFEB from the cytosol into the nucleus, through the nuclear pore (NP) by KPNB1/importinβ1. In the C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal lobar degeneration (ALS-FTD), the hexanucleotide (G4C2)RNA expansion (HRE) disrupts the nucleocytoplasmic transport of TFEB, compromising autophagy. Here we show that a molecular chaperone, the SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1), facilitates TFEB transport into the nucleus by chaperoning the NP protein (i.e., nucleoporin) POM121 which recruits KPNB1. In NSC34 cells, HRE reduces TFEB transport by interfering with the association between SIGMAR1 and POM121, resulting in reduced nuclear levels of TFEB, KPNB1, and the autophagy marker LC3-II. Overexpression of SIGMAR1 or POM121, or treatment with the highly selective and potent SIGMAR1 agonist pridopidine, currently in phase 2/3 clinical trials for ALS and Huntington disease, rescues all of these deficits. Our results implicate nucleoporin POM121 not merely as a structural nucleoporin, but also as a chaperone-operated signaling molecule enabling TFEB-mediated autophagy. Our data suggest the use of SIGMAR1 agonists, such as pridopidine, for therapeutic development of diseases in which autophagy is impaired. Abbreviations: ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementiaC9ALS-FTD, C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal dementiaCS, citrate synthaseER, endoplasmic reticulumGSS, glutathione synthetaseHRE, hexanucleotide repeat expansionHSPA5/BiP, heat shock protein 5LAMP1, lysosomal-associated membrane protein 1MAM, mitochondria-associated endoplasmic reticulum membraneMAP1LC3/LC3, microtubule-associated protein 1 light chain 3NP, nuclear poreNSC34, mouse motor neuron-like hybrid cell lineNUPs, nucleoporinsPOM121, nuclear pore membrane protein 121SIGMAR1/Sigma-1R, sigma non-opioid intracellular receptor 1TFEB, transcription factor EBTMEM97/Sigma-2R, transmembrane protein 97</p

Cholinergic system.

<p>Effects of oAβ_25–35 (10 µg/rat) icv injection on VAChT immunolabelling within the nucleus basalis of Meynert (A), mediobasal hypothalamus (B), parietal cortex (C) and hippocampus (D) determined in control untreated rats and 6 weeks after Aβ_25–35 injection. In (B): 3v: third ventricle. In (C): levels I to V cortical layers are indicated. In (D): brackets show the hippocampus granular cell layer. cc: corpus callosum. Scale bars  = 100 µm. Variations in VAChT levels in the hypothalamus (B) and hippocampus (D), determined in rats by western blot 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). VAChT (70 kDa) variations were normalized with β-tubulin (β-tub, 55 kDa) variations and compared with untreated rats (control group: C). The results are expressed as means ± SEM. *p<0.05 and **p<0.01 <i>vs</i>. control group, +p<0.05 and ++p<0.01 <i>vs</i>. scrambled treated rats. The number of animals in each group is indicated within the columns.</p

Hippocampus integrity.

<p>Variations in hippocampus pyramidal cell numbers determined in rats 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). <b>A.</b> Representative microphotographs of coronal sections of Cresyl violet stained hippocampus CA1, CA2, CA3 and DG subfields, obtained in control untreated rats and after scrambled Aβ_25–35 peptide or Aβ_25–35 icv injection. Scale bar  = 300 and 100 µm. <b>B.</b> Average numbers of hippocampus pyramidal cells determined in untreated control rats (C) and 6 weeks after icv injection of scrambled Aβ_25–35 peptide (10 µg/rat; negative control) or oAβ_25–35 (10 µg/rat). The results are expressed as means ± SEM (with n  = 4 per group). *p<0.05 and **p<0.01 vs. control rats, +p<0.05 and ++p<0.01 vs. respective scrambled peptide-treated rats. <b>C.</b> Effects of oAβ_25–35 (10 µg/rat) icv injection on hippocampus dendate gyrus (DG) neurogenesis using PSA-NCAM immunolabeling determined in untreated control rats and 6 weeks after Aβ_25–35 scrambled amyloid peptide (10 µg/rat; negative control) or oAβ_25–35 injection. Neurogenesis was visualized within coronal sections of the DG with Alexafluor555-labeled specific antibody against PSA-NCAM (red immunolabeling), while the nucleus was counterstained with DAPI (blue labeling). Scale bars = 200 µm.</p

Brain localization of Aβ_25–35 and particle characterization of Aβ_25–35 solutions A–I.

<p>Localization within brain structures of oAβ_25–35-HLF, determined 6 weeks after its icv injection (10 µg/rat). oAβ_25–35-HLF was visualized in green, while the nucleus was counterstained with DAPI (blue labeling). Abbreviations: 3V: third ventricle; alv: alveus of the hippocampus; CA1: field CA1 of hippocampus; CA3: field CA3 of the hippocampus; cc: corpus callosum; D3V: dorsal third ventricle; ec: external capsule; fi: fimbria of the hippocampus; FrA: frontal association cortex; hf: hippocampal fissure; LV: lateral ventricle; MEE: median eminence, external part; MEI: median eminence, internal part; MHb: medial habenular nucleus; PVN: paraventricular hypothalamic nucleus; PVP: paraventricular thalamic nucleus, posterior part. Arrowhead: blood vessel. Scale bar  = 100 µm. <b>J.</b> Particle size distribution of the different fractions of Aβ_25–35 solution (1 µg/µl) was determined by PCS at 25°C. Samples were prepared as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053117#s2" target="_blank">materials and methods</a> section. Black curve: Aβ_25–35 peptide dissolved in hexafluoroisopropanol (HFIP); Red curve: solution of aggregated Aβ_25–35 peptide; Green curve: supernatant of aggregated Aβ_25–35 peptide centrifuged at 1 000 g; Purple curve: re-suspended pellet of aggregated Aβ_25–35 peptide obtained after centrifugation at 1 000 g; Blue curve: supernatant of aggregated Aβ_25–35 peptide centrifuged at 16 000 g. Data were analyzed using a Zetasizer software 6.01 and expressed as size frequency distribution (%) in function of particles size (nm).</p