Organ Weights in NPC1 Mutant Mice Partly Normalized by Various Pharmacological Treatment Approaches.

Niemann-Pick Type C1 (NPC1, MIM 257220) is a rare, progressive, lethal, inherited autosomal-recessive endolysosomal storage disease caused by mutations in the NPC1 leading to intracellular lipid storage. We analyzed mostly not jet known alterations of the weights of 14 different organs in the BALB/cNctr-Npc1m1N/-J Jackson Npc1 mice in female and male Npc1+/+ and Npc1-/- mice under various treatment strategies. Mice were treated with (i) no therapy, (ii) vehicle injection, (iii) a combination of miglustat, allopregnanolone, and 2-hydroxypropyl-ß-cyclodextrin (HPßCD), (iv) miglustat, and (v) HPßCD alone starting at P7 and repeated weekly throughout life. The 12 respective male and female wild-type mice groups were evaluated in parallel. In total, 351 mice (176 Npc1+/+, 175 Npc1-/-) were dissected at P65. In both sexes, the body weights of None and Sham Npc1-/- mice were lower than those of respective Npc1+/+ mice. The influence of the Npc1 mutation and/or sex on the weights of various organs, however, differed considerably. In males, Npc1+/+ and Npc1-/- mice had comparable absolute weights of lungs, spleen, and adrenal glands. In Npc1-/- mice, smaller weights of hearts, livers, kidneys, testes, vesicular, and scent glands were found. In female Npc1-/- mice, ovaries, and uteri were significantly smaller. In Npc1-/- mice, relative organ weights, i.e., normalized with body weights, were sex-specifically altered to different extents by the different therapies. The combination of miglustat, allopregnanolone, and the sterol chelator HPßCD partly normalized the weights of more organs than miglustat or HPßCD mono-therapies.

Gender-Specific Effects of Two Treatment Strategies in a Mouse Model of Niemann-Pick Disease Type C1.

In a mouse model of Niemann-Pick disease type C1 (NPC1), a combination therapy (COMBI) of miglustat (MIGLU), the neurosteroid allopregnanolone (ALLO) and the cyclic oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (HPßCD) has previously resulted in, among other things, significantly improved motor function. The present study was designed to compare the therapeutic effects of the COMBI therapy with that of MIGLU or HPßCD alone on body and brain weight and the behavior of NPC1-/- mice in a larger cohort, with special reference to gender differences. A total of 117 NPC1-/- and 123 NPC1+/+ mice underwent either COMBI, MIGLU only, HPßCD only, or vehicle treatment (Sham), or received no treatment at all (None). In male and female NPC1-/- mice, all treatments led to decreased loss of body weight and, partly, brain weight. Concerning motor coordination, as revealed by the accelerod test, male NPC1-/- mice benefited from COMBI treatment, whereas female mice benefited from COMBI, MIGLU, and HPßCD treatment. As seen in the open field test, the reduced locomotor activity of male and female NPC1-/- mice was not significantly ameliorated in either treatment group. Our results suggest that in NPC1-/- mice, each drug treatment scheme had a beneficial effect on at least some of the parameters evaluated compared with Sham-treated mice. Only in COMBI-treated male and female NPC+/+ mice were drug effects seen in reduced body and brain weights. Upon COMBI treatment, the increased dosage of drugs necessary for anesthesia in Sham-treated male and female NPC1-/- mice was almost completely reduced only in the female groups.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

A therapy with miglustat, 2-hydroxypropyl-ß-cyclodextrin and allopregnanolone restores splenic cholesterol homeostasis in Niemann-pick disease type C1.

BACKGROUND: Niemann-Pick disease type C1 (NPC1) is an autosomal-recessive lipid-storage disorder with an estimated minimal incidence of 1/120,000 live births. Besides other neuronal and visceral symptoms, NPC1 patients develop spleen dysfunction, isolated spleno- or hepatosplenomegaly and infections. The mechanisms of splenomegaly and alterations of lipid metabolism-related genes in NPC1 disease are still poorly understood. METHODS: Here, we used an NPC1 mouse model to study a splenoprotective effect of a treatment with miglustat, 2-hydroxypropyl-ß-cyclodextrin and allopregnanolone and showed that this treatment has a positive effect on spleen morphology and lipid metabolism. RESULTS: Disease progress can be halted and blocked at the molecular level. Mutant Npc1 (Npc1-/-) mice showed increased spleen weight and increased lipid accumulation that could be avoided by our treatment. Also, FACS analyses showed that the increased number of splenic myeloid cells in Npc1-/- mice was normalized by the treatment. Treated Npc1-/- mice showed decreased numbers of cytotoxic T cells and increased numbers of T helper cells. CONCLUSIONS: In summary, the treatment promotes normal spleen morphology, stabilization of lipid homeostasis and blocking of inflammation, but alters the composition of T cell subtypes.

Fast and reliable dissection of porcine parathyroid glands - A protocol for molecular and histological analyses.

As calcium and phosphorus are of vital importance for life, physiological activity of the parathyroid glands (PTGs) is crucial to maintain mineral homeostasis and bone mineralization. However, PTG-specific molecular routes in response to environmental factors and intrinsic hormonal responses are not yet fully understood. Since nutrient requirements, pathophysiology and functional genomics of pigs are similar to those of humans, pigs might be a suitable model to study the holistic gene expression and physiological aspects of the parathyroid gland, which could be used in both animal sciences and biomedical research. However, due to their small size and hidden location, the dissection of the PTGs, particularly in pigs, is difficult. Therefore, a protocol for untrained dissectors has been established that allows a fast and reliable identification of the PTGs in domestic pigs. Based on their localization within the cranial thymus near the carotid bifurcation, sampling was verified by histological staining and mRNA expression pattern. Analyses revealed the prominence of parathyroid hormone (PTH)-producing chief cells. Moreover, the copy numbers of PTH differed substantially between the PTGs and their surrounding thymus tissue, as PTH was expressed virtually exclusively in the PTGs. The developed protocol will substantially facilitate a fast and reliable dissection of porcine PTGs which is essential for studies characterizing the molecular mechanisms of parathyroid glands, e.g. when applying new feeding strategies in pigs.

Evaluation of Two Liver Treatment Strategies in a Mouse Model of Niemann-Pick-Disease Type C1.

Niemann-Pick-disease type C1 (NPC1) is an autosomal-recessive cholesterol-storage disorder. Besides other symptoms, NPC1 patients develop liver dysfunction and hepatosplenomegaly. The mechanisms of hepatomegaly and alterations of lipid metabolism-related genes in NPC1 disease are still poorly understood. Here, we used an NPC1 mouse model to study an additive hepatoprotective effect of a combination of 2-hydroxypropyl-ß-cyclodextrin (HPßCD), miglustat and allopregnanolone (combination therapy) with the previously established monotherapy using HPßCD. We examined transgene effects as well as treatment effects on liver morphology and hepatic lipid metabolism, focusing on hepatic cholesterol transporter genes. Livers of Npc1-/- mice showed hepatic cholesterol sequestration with consecutive liver injury, an increase of lipogenetic gene expression, e.g., HMG-CoA, a decrease of lipolytic gene expression, e.g., pparα and acox1, and a decrease of lipid transporter gene expression, e.g., acat1, abca1 and fatp2. Both, combination therapy and monotherapy, led to a reduction of hepatic lipids and an amelioration of NPC1 liver disease symptoms. Monotherapy effects were related to pparα- and acox1-associated lipolysis/ß-oxidation and to fatp2-induced fatty acid transport, whereas the combination therapy additionally increased the cholesterol transport via abca1 and apoE. However, HPßCD monotherapy additionally increased cholesterol synthesis as indicated by a marked increase of the HMG-CoA and srebp-2 mRNA expression, probably as a result of increased hepatocellular proliferation.

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice.

Niemann-Pick Type C1 (NPC1) is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1nihNpc1-/- mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1-/- animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Intrastriatal injection of botulinum neurotoxin-A is not cytotoxic in rat brain - A histological and stereological analysis.

Parkinson's disease (PD) is caused by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in a deficiency of dopamine in the striatum and an increased release of acetylcholine by tonically active interneurons. Botulinum neurotoxin-A (BoNT-A) is well known for blocking transmitter release by cholinergic presynaptic terminals. Treating striatal hypercholinism by local application of BoNT-A could be a possible new local therapy option of PD. In previous studies of our group, we analyzed the effect of BoNT-A injection into the CPu of 6-OHDA lesioned hemiparkinsonian rats. Our studies showed that BoNT-A application in hemiparkinson rat model is capable of abolishing apomorphine induced rotations for approximately 3 months. Regularly occurring axonal swellings in the BoNT-A infiltrated striata were also discovered, which we named BoNT-A induced varicosities (BiVs). Résumé: Here we investigated the long-term effect of the injection of 1ng BoNT-A into the right CPu of naive Wistar rats on the number of ChAT-ir interneurons as well as on the numeric density and the volumetric size of the BiVs in the CPu. Significant differences in the number of ChAT-ir neurons between the right BoNT-A treated CPu and the left untreated CPu were not detected up to 12 month post BoNT-A injection. The numeric density of BiVs in the treated CPu reached a maximum 3 months after BoNT-A treatment and decreased afterwards, whereas the volume of single BiVs increased steadily throughout the whole time course of the experiment.

Reduced cerebellar neurodegeneration after combined therapy with cyclodextrin/allopregnanolone and miglustat in NPC1: a mouse model of Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disease characterized by a deficiency of NPC1 gene function. The malfunction of protein results in a progressive accumulation of lipids in many organs. A combined approach with substrate-reduction therapy (SRT) and byproduct therapy (BPT) has been shown to ameliorate the disease course in a mutant mouse model (NPC1(-/-)). The present study examines the morphological parameters underlying these changes. For the combined SRT/BPT treatment, NPC1(-/-) mutant mice (NPC1(-/-SRT/BPT)) were injected with allopregnanolone/cyclodextrin weekly, starting at postnatal day (P) 7. Starting at P10, a miglustat injection was administered daily until P23. Thereafter, miglustat was added to the powdered chow. For the sham treatment, both mutant NPC1(-/-) (NPC1(-/-sham)) and wild-type (NPC1(+/+sham)) mice received an NaCl injection and were fed powdered chow without miglustat. Analysis was performed on cerebellar slices by histology and immunohistochemistry. The volumes and cell counts of cerebellar structures were quantified. Additionally, ultrastructural analysis was performed with transmission electron microscopy. In agreement with previous studies, the current study demonstrates Purkinje cell degeneration in the mutant mice, which was partially abrogated by SRT/BPT. The volumes of cerebellar white matter and molecular layer were reduced as well. Also, the number of neurons was reduced in granular and molecular layers. However, only the molecular layer benefited from the therapy, as shown by an increase in the volume and the amount of neurons. The volume and number of neurons of the deep cerebellar nuclei were significantly decreased in mutant mice; an appreciable therapeutic benefit could be demonstrated for the nucleus interpositus.

Corneal alterations during combined therapy with cyclodextrin/allopregnanolone and miglustat in a knock-out mouse model of NPC1 disease.

BACKGROUND: Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in the NPC1 gene, which result in accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system as well as limiting membranes. We have previously shown the corneal involvement in NPC1 pathology in form of intracellular inclusions in epithelial cells and keratocytes. The purpose of the present study was to clarify if these inclusions regress during combined substrate reduction- and by-product therapy (SRT and BPT). METHODOLOGY/PRINCIPAL FINDINGS: Starting at postnatal day 7 (P7) and thereafter, NPC1 knock-out mice (NPC1(-/-)) and wild type controls (NPC1(+/+)) were injected with cyclodextrin/allopregnanolone weekly. Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. The sham group was injected with 0.9% NaCl at P7, thereafter daily starting at P10 until P23, and fed powdered chow starting at P23. For corneal examination, in vivo confocal laser-scanning microscopy (CLSM) was performed one day before experiment was terminated. Excised corneas were harvested for lipid analysis (HPLC/MS) and electron microscopy. In vivo CLSM demonstrated a regression of hyperreflective inclusions in all treated NPC1(-/-)mice. The findings varied between individual mice, demonstrating a regression, ranging from complete absence to pronounced depositions. The reflectivity of inclusions, however, was significantly lower when compared to untreated and sham-injected NPC1(-/-) mice. These confocal findings were confirmed by lipid analysis and electron microscopy. Another important CLSM finding revealed a distinct increase of mature dendritic cell number in corneas of all treated mice (NPC1(-/-) and NPC1(+/+)), including sham-treated ones. CONCLUSIONS/SIGNIFICANCE: The combined substrate reduction- and by-product therapy revealed beneficial effects on the cornea. In vivo CLSM is a non-invasive tool to monitor disease progression and treatment effects in NPC1 disorder.

Decreased expression of myelin gene regulatory factor in Niemann-Pick type C 1 mouse.

Niemann-Pick type C 1 (NPC1) disease is an autosomal recessive cholesterol transport defect resulting in a neurodegenerative process in patients mainly at an early age, although some patients may start with manifestation in adult. Since loss of myelin is considered as a main pathogenetic factor, the precise mechanism inducing dysmylination in NPC1 disease is still unclear. In the present study, a quantitative evaluation on the myelin protein and its regulatory factors of oligodendrocytes, such as SRY-related HMG-box 10 (Sox10), Yin Yang 1 factor (YY1) and myelin gene regulatory factor (MRF), in different parts of the brain and spinal cord was performed in NPC1-mutant mice. The results showed that NPC1 protein was expressed in oligodendrocytes and the amount of myelin protein was generally decreased in all parts of the brain and spinal cord in NPC1-mutant mice. Compared to wild type, the amount of Sox10 and YY1 was not different in NPC1-mutant mice, but MRF was significantly decreased, suggesting a possible mechanism perturbing differentiation of oligodendrocytes and the myelination process in the NPC1-mutant mouse.

Efferent projections of the anterior and posterodorsal regions of the medial nucleus of the amygdala in the mouse.

The efferent projections of the anterior and posterodorsal part of the medial nucleus (MePD) in the mouse were studied by means of anterograde axonal tracing using biotinylated dextran amine. The MePD axons ran mainly via the stria terminalis and to a lesser extent via the ventral amygdalofugal pathway. The projections to the forebrain were broadly distributed and varied from very strong to scant. The most significant connections were destined to the bed nucleus of the stria terminalis in which all parts of the medial division were innervated by MePD neurons. Moderate projections reached the limbic striatum (nucleus accumbens), olfactory tubercle and the lateral septal nucleus. The substantia innominata was also innervated by the MePD, and especially the projection to its ventral portion was substantial. The profuse innervation of the medial preoptic nucleus and medial preoptic area indicated significant involvement of the MePD in sexual behavior. Many hypothalamic nuclei were innervated but to a different extent. The very strong innervation of the ventral premammillary nucleus further indicated the involvement of the MePD in the neuronal circuitry for sexual behavior. Substantial projections also reached the anterior hypothalamus and tuber cinereum, while the connections to the lateral hypothalamus were widespread but showed moderate density. MePD strongly innervated the ventrolateral part of the ventromedial hypothalamic nucleus and moderately its remaining parts. The neurosecretory hypothalamic nuclei and the arcuate nucleus contained only a few MePD terminals. The thalamic innervation was very scant and reached the lateral habenular nucleus and the nuclei of the midline. The mesencephalic connections were moderate to sparse and projected to the mesolimbic dopaminergic groups in the ventral tegmental area, the pars lateralis and the dorsal tier of the substantia nigra pars compacta, the periaqueductal gray and the dorsal raphe nucleus. The present results principally resembled data known in other rodent species; however, the efferents of the MePD often differed in extent and/or topical distribution.

Ciliary neurotrophic factor overexpression in neural progenitor cells (ST14A) increases proliferation, metabolic activity, and resistance to stress during differentiation.

Neurotrophic factors exert considerable neuroprotective and neurorestorative effects in neurodegenerative diseases. Because neuronal progenitor cells have, at least in part, the potency to restore degenerated neuronal networks, transgenic high-dosage expression of neurotrophins by these cells in neurotransplantation may be advantageous. In the present study, a retroviral vector containing the gene of rat ciliary neurotrophic factor (rCNTF) was permanently transfected into a striatal neuronal progenitor cell line. Qualitative and quantitative analyses demonstrated a sustained expression of the transgene; i.e., rCNTF was present at the mRNA level and protein level. Moreover, cocultivation in separate chambers of transgenic CNTF-ST14A cells and CNTF-dependent TF1 cells exerted typical biological effects, such as increased proliferation and differentiation of the TF1 cells, indicating the functional integrity of the secreted recombinant neurotrophin. The CNTF-ST14A cells displayed improved stress response compared with native ST14A cells under differentiation conditions, i.e., at the nonpermissive temperature of 39 degrees C and after staurosporine exposure, respectively. This effect coincided with a relatively reduced apoptosis rate and a raised metabolic activity of CNTF-ST14A cells at 39 degrees C. Neurotransplantation of CNTF-ST14A cells in the rat quinolinic acid model of Huntington's disease showed a significant and sustained decline in pathological apomorphine-induced rotations compared with parental ST14A cells. We conclude that sustained functional transgene CNTF production improves stress response as well as metabolic activity, making CNTF-ST14A cells a promising tool for neurotransplantation in the quinolinic acid model of Huntington's disease.