Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development.

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

Expression pattern of Nav2 in the murine CNS with development.

Neuron navigator 2 (NAV2, RAINB1, POMFIL2, HELAD1, unc53H2) is essential for nervous system development. In the present study the spatial distribution of Nav2 transcript in mouse CNS during embryonic, postnatal and adult life is examined. Because multiple NAV2 proteins are predicted based on alternate promoter usage and RNA splicing, in situ hybridization was performed using probes designed to the 5' and 3' ends of the Nav2 transcript, and PCR products using primer sets spanning the length of the mRNA were also examined by real time PCR (qPCR). These studies support full-length Nav2 transcript as the predominant form in the wild-type mouse CNS. The developing cortex, hippocampus, thalamus, olfactory bulb, and granule cells (GC) within the cerebellum show the highest expression, with a similar staining pattern using either the 5'Nav2 or 3'Nav2 probe. Nav2 is expressed in GC precursors migrating over the cerebellar primordium as well as in the postmitotic premigratory cells of the external granule cell layer (EGL). It is expressed in the cornu ammonis (CA) and dentate gyrus (DG) throughout hippocampal development. In situ hybridization was combined with immunohistochemistry for Ki67, CTIP2 and Nissl staining to follow Nav2 transcript location during cortical development, where it is observed in neuroepithelial cells exiting the germinal compartments, as well as later in the cortical plate (CP) and developing cortical layers. The highest levels of Nav2 in all brain regions studied are observed in late gestation and early postnatal life which coincides with times when neurons are migrating and differentiating. A hypomorphic mouse that lacks the full-length transcript but expresses shorter transcript shows little staining in the CNS with either probe set except at the base of the cerebellum, where a shorter Nav2 transcript is detected. Using dual fluorescent probe in situ hybridization studies, these cells are identified as oligodendrocytes and are detected using both Olig1 and the 3'Nav2 probe. The identification of full-length Nav2 as the primary transcript in numerous brain regions suggests NAV2 could play a role in CNS development beyond that of its well-established role in the cerebellum.

A New 1,25 Dihydroxy Vitamin D Analog with Strong Bone Anabolic Activity in OVX Rats with Little or no Bone Resorptive Activity.

A new 1α,25-dihydroxy vitamin D3 analog (2-methylene-22(E)-(24R)-22-dehydro-1α,24,25-trihydroxy-19-norvitamin D3 or WT-51) has been tested as a possible therapeutic for osteoporosis. It is 1/10th as active as 1,25(OH)2 D3 in binding affinity for the vitamin D receptor but is at least 200 times more active than 1,25(OH)2 D3 and equal to that of 2MD (2-methylene-19-nor-(20S)-1α,25(OH)2 D3 , an analog previously tested in postmenopausal women), in supporting bone formation by isolated osteoblasts in culture. However, in contrast to 2MD, it is virtually inactive on bone resorption in vivo. WT-51 markedly increased bone mass (lumbar and femur) in ovariectomized (OVX) female rats. Further, bone strength tested by the three-point bending system is significantly increased by WT-51. Thus, WT-51 is an attractive candidate for the treatment of postmenopausal osteoporosis. © 2019 American Society for Bone and Mineral Research.

4-HPR Is an Endoplasmic Reticulum Stress Aggravator and Sensitizes Breast Cancer Cells Resistant to TRAIL/Apo2L.

BACKGROUND/AIM: N-(4-hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid, less toxic than the parent all-trans retinoic acid (RA). Unlike RA, 4-HPR induces apoptosis in tumor cells. Because 4-HPR can hydrolyze to liberate RA, a potent human teratogen, the unhydrolyzable ketone analog of 4-HPR, 4-hydroxybenzylretinone (4-HBR) has been prepared and has been found to cause apoptosis in tumor cells and shrink carcinogen-induced rat mammary tumors as 4-HPR does. Herein, we examined the mechanism whereby 4-HPR and 4-HBR induce apoptosis and death in breast cancer cells. MATERIALS AND METHODS: Gene expression profiling was conducted in MCF-7 cells over a 1.5- to 6-h time course and changes were validated by quantitative polymerase chain reaction (qPCR). Growth arrest and DNA damage-inducible protein 153 (GADD153 or C/EBP homologous protein, CHOP) was knocked down and the effect on 4-HPR-induced cell death and gene expression was assessed. 4-HPR synergy with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL or Apo2 ligand) was also examined. RESULTS: Drug treatment induced increased expression of endoplasmic reticulum (ER) stress-related and pro-apoptotic genes. Gene expression changes were verified by qPCR in three invasive ductal breast carcinoma cell lines (MCF-7, T-47D, MDA-MB-231). GADD153 showed the largest increase in the microarray experiment; however, knockdown of GADD153 did not abrogate apoptosis and death. Genes related to the extrinsic pathway of apoptosis including a receptor for TRAIL, death receptor 5 (DR5), were up-regulated by drug treatment. A dose of 4-HPR that alone is ineffective in killing TRAIL-resistant MCF-7 cells, synergized with recombinant TRAIL to induce breast cancer cell death. CONCLUSION: 4-HPR and analogs might be useful in sensitizing tumor cells to death receptor agonists.

Differential activity of 2-methylene-19-nor vitamin D analogs on growth factor gene expression in rhino mouse skin and comparison to all-trans retinoic acid.

While all 2-methylene-19-nor analogs of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) tested produce an increase in epidermal thickness in the rhino mouse, only a subset reduce utricle size (comedolysis). All-trans retinoic acid (atRA) also causes epidermal thickening and a reduction in utricle size in the rhino mouse. We now report that 2-methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol (2MbisP), a comedolytic analog, increases epidermal thickening more rapidly than does atRA, while both reduce utricle area at an equal rate. Whereas unlike atRA, 2MbisP does not alter the epidermal growth factor receptor ligand, heparin-binding epidermal growth factor-like growth factor, it does increase the expression of both amphiregulin and epigen mRNA, even after a single dose. In situ hybridization reveals an increase in these transcripts throughout the closing utricle as well as in the interfollicular epidermis. The mRNAs for other EGFR ligands including betacellulin and transforming growth factor-α, as well as the epidermal growth factor receptor are largely unaffected by 2MbisP. Another analog, 2-methylene-19-nor-(20S)-26,27-dimethylene-1α,25-dihydroxyvitamin D3 (CAGE-3), produces epidermal thickening but fails to reduce utricle size or increase AREG mRNA levels. CAGE-3 modestly increases epigen mRNA levels, but only after 5 days of dosing. Thus, 2-MbisP produces unique changes in epidermal growth factor receptor ligand mRNAs that may be responsible for both epidermal proliferation and a reduction in utricle size.

Pharmacokinetics of a New Oral Vitamin D Receptor Activator (2-Methylene-19-Nor-(20S)-1α,25-Dihydroxyvitamin D3) in Patients with Chronic Kidney Disease and Secondary Hyperparathyroidism on Hemodialysis.

BACKGROUND: 2-Methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (DP001 or 2MD) is a novel, potent 1α-hydroxylated vitamin D analog that binds to the vitamin D receptor and suppresses parathyroid hormone synthesis and secretion with potential for an improved safety profile compared to existing active vitamin D analogs. The purpose of this study was to evaluate the pharmacokinetics of DP001 given orally after hemodialysis. METHODS: DP001 (550 ng) was given orally to 11 hemodialysis patients with secondary hyperparathyroidism after each dialysis session (3 times/week) for 4 weeks. Pharmacokinetic analyses were performed after the first and final dose. RESULTS: After the first and final dose, the half-life of DP001 was similar (55.8 ± 13.0 and 50.8 ± 8.2 h, respectively). At 4 weeks, the time to maximum plasma concentration was 4.0 ± 0.8 h, with a concentration maximum of 3.4 ± 0.3 pg/mL. The area under the curve (0 to infinity) after the final dose was 204.3 ± 23.9 pg h/mL, and apparent volume of distribution was 2.03 ± 0.22 L/kg. At week 4, mean intact parathyroid hormone was suppressed 33% from the baseline (pre-dose) value (313 ± 52 vs 462 ± 39 pg/mL, respectively). No clinically significant changes from baseline values were found for vital signs, electrocardiogram measurements, or other laboratory parameters, including serum calcium and phosphorus. CONCLUSIONS: In hemodialysis patients, DP001 has a longer half-life than existing vitamin D therapies and enables control of parathyroid hormone when administered every 2-3 days on the day of dialysis. It is effective at a lower concentration maximum and area under the curve than other clinically available vitamin D compounds. DP001 may represent a therapeutic improvement over existing compounds due to rapid and extensive distribution to its target and its long half-life enabling sustained parathyroid hormone suppression. These studies support further evaluation of DP001 in longer-term treatment of secondary hyperparathyroidism.

2MD (DP001), a Single Agent in the Management of Hemodialysis Patients: A Randomized Trial.

BACKGROUND: Vitamin D analogs and calcimimetics are used to manage secondary hyperparathyroidism (SHPT) in dialysis patients. DP001 is an oral vitamin D analog that suppresses parathyroid hormone (PTH) in uremic rats, osteopenic women, and hemodialysis patients. The safety and effectiveness of DP001 suppressing PTH in dialysis patients previously managed with active vitamin D with or without a calcimimetic are presented. METHODS: A multicenter, randomized, double-blind study compared DP001 to placebo in hemodialysis patients with serum-intact PTH (iPTH) ≥300 pg/ml. The primary efficacy endpoint was the proportion of patients achieving 2 consecutive ≥30% decreases in iPTH levels during the 12 weeks of treatment. Calcium, phosphorus, calcium × phosphorus product and safety were also evaluated. The responses to DP001 were compared in patients previously treated with both active vitamin D and a calcimimetic to those previously on active vitamin D alone. RESULTS: Sixty-two patients were randomized (n = 34 DP001; n = 28 placebo). At week 12, 78% of all DP001-treated patients and 7% of all placebo-treated patients achieved the primary endpoint (p < 0.0001); iPTH fell 45% in the DP001 group and increased 37% in the placebo group. No patient exceeded the safety threshold of 2 consecutively corrected serum calcium levels ≥11.0 mg/dl. Patients previously on cinacalcet plus active vitamin D also responded to DP001 (n = 10) resulting in a 55% decrease in iPTH, while those on placebo (n = 9) increased by 70%. CONCLUSION: DP001 safely and effectively suppressed iPTH in hemodialysis patients with SHPT that were previously managed with active vitamin D alone or with a calcimimetic (www.clinicaltrials.gov, NCT01922843).

Use of 2MD, a Novel Oral Calcitriol Analog, in Hemodialysis Patients with Secondary Hyperparathyroidism.

BACKGROUND: Use of existing therapies for secondary hyperparathyroidism (SHPT), such as calcitriol or paricalcitol, is frequently limited by the development of hypercalcemia. 2-Methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD; DP001) is a novel and a more potent vitamin D receptor activator (VDRA) that more selectively localizes in the parathyroid gland, and has a wider therapeutic margin in the uremic rat model than calcitriol and paricalcitol. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Hemodialysis patients were enrolled and dosed with 110, 220, 330, 440, or 550 ng of 2MD orally thrice weekly for 4 weeks. Responders were defined as patients having a ≥30% reduction in parathyroid hormone (PTH) from baseline, and were assessed at weeks 2 and 4. RESULTS: Of 31 patients recruited, 24 completed the 4-week treatment. There was little or no reduction in PTH in the 110 and 220 ng dose cohorts. Higher dose cohorts had greater PTH suppression with more than half the patients in the 440 and 550 ng dose cohorts considered responders (≥30% PTH reduction from baseline). None had oversuppression of PTH or hypercalcemia (corrected serum calcium >10.6 mg/dl). Plasma drug concentration increased with increasing dose, and all responders achieved a 2MD concentration of ≥1.5 pg/ml. All dose levels of 2MD were well tolerated without safety concerns. CONCLUSIONS: In hemodialysis patients with SHPT, 2MD, at thrice weekly oral doses of 440 and 550 ng, is well tolerated and effectively suppresses PTH without hypercalcemia. Future studies are needed to study the long-term implications of treating ESRD patients with this novel VDRA.

Improved Synthesis of the C-Glucuronide/Glycoside of 4-Hydroxybenzylretinone (4-HBR).

Improvements in the synthesis of carbon-linked glucuronide/glucoside conjugates of cancer chemopreventive retinoids have been achieved starting with 2,3,4,6-tetra-O-benzyl-D-glucopyranose. The revised approach demonstrates better yields, eliminates the use of an expensive, carcinogenic protecting group reagent, and avoids much painstaking chromatography. The new approach should allow synthesis of larger quantities of the agents for detailed animal and mechanistic studies.

A complex RARE is required for the majority of Nedd9 embryonic expression.

Neural precursor cell expressed, developmentally down-regulated 9 (Nedd9, Casl, Hef1, p105cas, Ef1) is a scaffolding protein that assembles complexes involved in regulating cell adhesion, migration, division, and survival. Nedd9 is found very early in the developing embryonic nervous system. A highly conserved complex retinoic acid response element (RARE) is located 485 base pairs (bp) upstream of exon 2B in the promoter of the Nedd9 gene. Mice transgenic for a 5.2 kilobase (kb) region of the 2B Nedd9 promoter containing the RARE upstream of a lacZ reporter gene [Nedd9(RARE)-lacZ] show a large subset of the normal endogenous Nedd9 expression including that in the caudal hindbrain neuroepithelium, spinal cord, dorsal root ganglia (drg) and migrating neural crest (ncc). However, the transgenic mice do not recapitulate the native Nedd9 expression pattern in presumptive rhombomeres (pr) 3 and 5 of the early hindbrain, the base of the neuroepithelium in the midbrain, nor the forebrain telencephalon. Thus, the 5.2 kb region containing the intact RARE drives a large subset of Nedd9 expression, with additional sequences outside of this region needed to define the full complement of expression. When the 5.2 kb construct is modified (eight point mutations) to eliminate responsiveness of the RARE to all-trans retinoic acid (atRA) [Nedd9(mutRARE)-lacZ], virtually all ß-galactosidase (ß-gal, lacZ) expression is lost. Exposure of Nedd9(RARE)-lacZ transgenic embryos to excess atRA at embryonic day 8.0 (E8.0) leads to rostral ectopic transgene expression within 6 h whereas the Nedd9(mutRARE)-lacZ mutant does not show this effect. Thus the RARE upstream of the Nedd9 2B promoter is necessary for much of the endogenous gene expression during early development as well as ectopic expression in response to atRA.

Novel, selective vitamin D analog suppresses parathyroid hormone in uremic animals and postmenopausal women.

BACKGROUND: The use of 1α-hydroxylated vitamin D therapy to control secondary hyperparathyroidism in renal failure patients has been a success story, culminating with the demonstration of increased life expectancy in patients treated with these compounds. However, hypercalcemic episodes have been a recurrent problem with these therapies and have resulted in the added use of calcium mimetics. Clearly there is good reason to search for improved vitamin D therapy. In our inventory of vitamin D compounds, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) surfaced as a potential candidate. This was based on its preferential localization in the parathyroid gland and a clear suppression of serum parathyroid hormone (PTH) levels without a change in serum calcium in a clinical trial in postmenopausal women. METHODS: 2MD has now been tested in the rat 5/6-nephrectomy model of renal failure, and in postmenopausal women to determine if it can suppress serum PTH at doses that do not elevate serum calcium and serum phosphorus concentrations. RESULTS: Daily oral treatment of uremic rats on 2.5 ng/bw/day of 2MD dramatically suppressed PTH without a change in serum calcium or serum phosphorus. Further, PTH was suppressed in postmenopausal women after only 3 daily oral doses of 2MD that continued for 4 weeks with no change in serum calcium or serum phosphorus. CONCLUSION: These results coupled with a pharmacokinetic half-life of ~24 h suggest that 2MD given either daily or at the time of dialysis may be a superior therapy for secondary hyperparathyroidism in chronic renal failure patients.

26-Desmethyl-2-methylene-22-ene-19-nor-1α,25-dihydroxyvitamin D3 compounds selectively active on intestine.

Six new analogs of 2-methylene-19-nor-1α,25-dihydroxyvitamin D3, 6-7 and 8a,b-9a,b, have been synthesized. All compounds are characterized by a trans double bond located in the side chain between C-22 and C-23. While compounds 6 and 7 possess C-26 and C-27 methyls, compounds 8a,b and 9a,b lack one of these groups. A Lythgoe-based synthesis, employing the Wittig-Horner reaction was used for these preparations. Two different types of Δ(22)E-25-hydroxy Grundmann's ketone, having either only one stereogenic center located at position C-20 (20 and 21), or two stereogenic centers located at 20- and 25-positions (24a,b-25a,b) were obtained by a multi-step procedure from commercial vitamin D2. The introduction of a double bond at C-22 appeared to lower biological activity in vitro and in vivo. Further removal of a 26-methyl in these analogs had little effect on receptor binding, HL-60 differentiation and CYP24A expression but markedly diminished or eliminated in vivo activity on bone calcium mobilization while retaining activity on intestinal calcium transport.

14-3-3ε and NAV2 interact to regulate neurite outgrowth and axon elongation.

Neuron navigator 2 (NAV2) is required for all-trans retinoic acid (atRA) to induce neurite outgrowth in human neuroblastoma cells. Further, ectopic overexpression of full-length human NAV2 rescues an axonal elongation defect in the Caenorhabditis elegans unc-53 (NAV2 ortholog) mutant. Using a region of NAV2 that independently associates with the cytoskeleton as bait in a yeast-two-hybrid screen, 14-3-3ε was identified as a novel NAV2 interacting partner. Amino acids 761-960 of NAV2 are sufficient to confer a positive interaction with 14-3-3ε as evidenced by a two-hybrid screen and co-immunoprecipitation assay. Knockdown of 14-3-3ε leads to a decrease in atRA-mediated neurite outgrowth, similar to the elongation defects observed when NAV2 is depleted or mutated. Likewise, posterior lateral microtubule (PLM) defects in C. elegans fed unc-53 RNAi are similar to those fed ftt-2 (14-3-3 homolog) RNAi. The discovery of an interaction between NAV2 and 14-3-3ε could provide insight into the mechanism by which NAV2 participates in promoting cell migration and neuronal elongation.

26- and 27-Methyl groups of 2-substituted, 19-nor-1α,25-dihydroxylated vitamin D compounds are essential for calcium mobilization in vivo.

Twelve new analogs of 19-nor-1α,25-dihydroxyvitamin D36-17, were prepared by a multi-step procedure from known alcohols 18 and 19. We have examined the influence of removing two methyl groups located at C-25, as well as the 25-hydroxy group, on the biological in vitro and in vivo biological activity. Surprisingly, removal of the 26- and 27-methyl groups from either the 2α-methyl or 2-methylene-19-nor-1α,25-dihydroxyvitamin D3 reduced vitamin D receptor binding, HL-60 differentiation, and 25-hydroxylase transcription in vitro only slightly to moderately (compounds 6-13). However, these compounds were devoid of in vivo bone mobilization activity and had markedly reduced activity on intestinal calcium transport. The analogs 14-17 with a 2ß-methyl substitution had little or no activity in vitro and in vivo as expected from previous work.

Astrocytes as a regulated source of retinoic acid for the brain.

Retinaldehyde dehydrogenases (RALDH) catalyze the synthesis of the regulatory factor retinoic acid (RA). Cultured astrocytes express several of the RALDH enzyme family, and it has been assumed that this can be extrapolated to astrocytes in vivo. However, this study finds that few astrocytes in the rodent brain express detectable RALDH enzymes, and only when these cells are grown in culture are these enzymes upregulated. Factors controlling the expression of the RALDHs in cultured astrocytes were explored to determine possible reasons for differences between in vitro versus in vivo expression. Retinoids were found to feedback to suppress several of the RALDHs, and physiological levels of retinoids may be one route by which astrocytic RALDHs are maintained at low levels. In the case of RALDH2, in vivo reduction of vitamin A levels in rats resulted in an increase in astrocyte RALDH2 expression in the hippocampus. Other factors though are likely to control RALDH expression. A shift in astrocytic RALDH subcellular localization is a potential mechanism for regulating RA signaling. Under conditions of vitamin A deficiency, RALDH2 protein moved from the cytoplasm to the nucleus where it may synthesize RA at the site of the nuclear RA receptors. Similarly, in conditions of oxidative stress RALDH1 and RALDH2 moved from the cytoplasm to a predominantly nuclear position. Thus, the RALDHs have been revealed to be dynamic in their expression in astrocytes where they may maintain retinoid homeostasis in the brain.

A 20S combined with a 22R configuration markedly increases both in vivo and in vitro biological activity of 1α,25-dihydroxy-22-methyl-2-methylene-19-norvitamin D3.

Six new analogues of 1α,25-dihydroxy-19-norvitamin D(3) (3a-4b, 5, and 6) were prepared by a convergent synthesis applying the Wittig-Horner reaction as a key step. The influence of methyl groups at C-22 on their biological activity was examined. It was established that both in vitro and in vivo activity is strongly dependent on the configuration of the stereogenic centers at C-20 and C-22. Introduction of the second methyl group at C-22 (analogues 5 and 6) generates the compounds that are slightly more potent than 1α,25-(OH)(2)D(3) in the in vitro tests but much less potent in vivo. The greatest in vitro and in vivo biological activity was achieved when the C-20 is in the S configuration and the C-22 is in the R configuration. The building blocks for the synthesis, the respective (20R,22R)-, (20R,22S)-, (20S,22R)-, and (20S,22S)-diols, were obtained by fractional crystallization of mixtures of the corresponding diastereomers. Structures and absolute configurations of the diols 21a, 21b, and 22a as well as analogues 3a, 5, and 6 were confirmed by the X-ray crystallography.

Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase.

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)(2)D(3) analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner-Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80-1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3), further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)(2)D(3) and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.

The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells.

The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21(Cip1), a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G(1)/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation.

4-Hydroxybenzyl modification of the highly teratogenic retinoid, 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB), yields a compound that induces apoptosis in breast cancer cells and shows reduced teratogenicity.

Retinoids are a class of compounds with structural similarity to vitamin A. These compounds inhibit the proliferation of many cancer cell lines but have had limited medical application as they are often toxic at therapeutic levels. Efforts to synthesize retinoids with a greater therapeutic index have met with limited success. 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB) is one of the most biologically active all-trans-retinoic acid (atRA) analogues and is highly teratogenic. In this study, we show that modification of the TTNPB carboxyl group with an N-(4-hydroxyphenyl)amido (4HPTTNPB) or a 4-hydroxybenzyl (4HBTTNPB) group changes the activity of the compound in cell culture and in vivo. Unlike TTNPB, both compounds induce apoptosis in cancer cells and bind poorly to the retinoic acid receptors (RARs). Like the similarly modified all-trans-retinoic acid (atRA) analogues N-(4-hydroxyphenyl)retinamide (4-HPR/fenretinide) and 4-hydroxybenzylretinone (4-HBR), 4HBTTNPB is a potent activator of components of the ER stress pathway. The amide-linked analogue, 4HPTTNPB, is less toxic to developing embryos than the parent TTNPB, and most significantly, the 4-hydroxybenzyl-modified compound (4HBTTNPB) that cannot be hydrolyzed in vivo to the parent TTNPB compound is nearly devoid of teratogenic liability.

Nav2 hypomorphic mutant mice are ataxic and exhibit abnormalities in cerebellar development.

Development of the cerebellum involves a coordinated program of neuronal process outgrowth and migration resulting in a foliated structure that plays a key role in motor function. Neuron navigator 2 (Nav2) is a cytoskeletal-interacting protein that functions in neurite outgrowth and axonal elongation. Herein we show that hypomorphic mutant mice lacking the full-length Nav2 transcript exhibit ataxia and defects in cerebellar development. At embryonic day (E)17.5, the mutant cerebellum is reduced in size and exhibits defects in vermal foliation. Reduction in cell proliferation at early times (E12.5 and E14.5) may contribute to this size reduction. The full-length Nav2 transcript is expressed in the premigratory zone of the external granule layer (EGL). Granule cells in the germinal zone of the EGL appear to proliferate normally, however, due to the reduction in cerebellar circumference there are fewer total BrdU-labeled granule cells in the mutants, and these fail to migrate normally toward the interior of the cerebellum. In Nav2 hypomorphs, fewer granule cells migrate out of cerebellar EGL explants and neurite outgrowth from both explants and isolated external granule cell cultures is reduced. This suggests that the formation of parallel axon fibers and neuronal migration is disrupted in Nav2 mutants. This work supports an essential role for full-length Nav2 in cerebellar development, including axonal elongation and migration of the EGL neurons.