Altered functional properties of a TRPM2 variant in Guamanian ALS and PD

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism–dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca2+ and Mg2+, coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2P1018L, produces a missense change in the channel protein whereby proline 1018 (Pro1018) is replaced by leucine (Leu1018). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states

Cryopreservation of human mucosal tissues.

BackgroundCryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.Methods and findingsTo find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.ConclusionsCryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar

Cryopreservation of human mucosal tissues

<div><p>Background</p><p>Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.</p><p>Methods and findings</p><p>To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated “cryopreservation”) and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol (“vitrification”). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59–84%]) than before (50% [38–62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.</p><p>Conclusions</p><p>Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.</p></div

Cryopreserved and vitrified vaginal explants maintain similar tissue structure to fresh explants.

<p>Example images from one tissue donor of hematoxylin-and-eosin-stained sections of vaginal tissue after no treatment (left), cryopreservation (middle), and vitrification (right) (total n = 5 tissue donors). Scale bar indicates size (“μm”, micrometer).</p

Cryopreservation of intact colorectal tissue leads to greater cell numbers than cryopreservation of cell suspensions, while cellular functionality is similar.

<p><b>A</b>, Viability by trypan blue exclusion of colorectal cells digested from biopsies either fresh, cryopreserved as biopsies, or cryopreserved after digestion (n = 18 tissue donors). <b>B</b>, Live cell yield by trypan blue exclusion. <b>C</b>, Recovery of live cells after cryopreservation relative to fresh samples from the same donor. <b>D</b>, Background-subtracted cytokine production from colorectal T cells after stimulation with cytomegalovirus, Epstein-Barr virus and influenza virus peptides (CEF); HIV Gag peptides (Gag); phorbol 12-myristate 13-acetate and ionomycin (PMA/Iono); or staphylococcal enterotoxin B (SEB). Small gray symbols indicate individual samples, with gray lines connecting samples from the same tissue donors. Colored symbols show the mean across all samples from HIV⁺ (orange) and HIV^- (green) donors, with vertical lines showing the 95% confidence interval of the mean. <b>E</b>, Polyfunctionality of colorectal responses to stimulation. Each sub-bar corresponds to the percent of cells with the indicated number of functions, averaged across the donors. Total bar height indicates the total percent of cells with one or more functions.</p

Cryopreserved and vitrified vaginal explants show similar levels of metabolic activity to fresh explants.

<p><b>A</b>, Background-subtracted alamar blue fluorescence of vaginal explants after 5h of culture following cryopreservation and vitrification. “Wet” explants were immersed in vitrification medium during freezing and “dry” explants were blotted dry before freezing (n = 1 tissue donor). “NA” stands for “not applicable” and indicates non-vitrification conditions; “PBS”, “phosphate buffered saline”; “bg”, “background.” <b>B</b>, Normalized alamar blue fluorescence of vaginal explants (expressed as percent of the fluorescence measured in the fresh explants from the same donor). Promising conditions from A were repeated with explants from 3 tissue donors, which are shown together with the data from the first tissue donor in A. <b>C</b>, Normalized alamar blue fluorescence of vaginal explants comparing vitrification with aluminum or copper foil (n = 3 tissue donors). <b>D</b>, Summary of experiments comparing cell metabolic activity after cryopreservation and vitrification (aluminum foil) in vaginal explants (n = 10 tissue donors). <b>E</b>, Effect of tissue size on cell metabolic activity after cryopreservation or vitrification (aluminum foil). Here fluorescence was normalized to the fresh explants of the same diameter (n = 1 tissue donor; “mm”, millimeter). In A and E, symbols indicate the average of two to three explants. In B-D, smaller gray points indicate the average of duplicate explants, with gray lines indicating samples from the same tissue donor. Black points show the mean across all tissue donors and black vertical lines show the 95% confidence interval of the mean.</p