Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-stan... more Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-standard method for analyzing modifications in gene expression in cells and tissues. However, large quantities of high-quality RNA samples are needed for analyzing the expression of multiple genes from one human tissue sample. Here, we provide an optimized protocol for extracting large amounts of RNA from human nasal mucosal biopsies. The quality and quantity of samples were sufficient for qRT-PCR analyses of the expressions of various genes, in duplicate. In contrast to other proto
Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is ... more Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD anima...
Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson’s disease (PD) in hu... more Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson’s disease (PD) in humans. The first central relay of the olfactory pathway, the olfactory bulb (OB), depends, among other things, on an intact, functional crosstalk between dopaminergic interneurons and dopamine receptors (D2/D3R). In rats, hemiparkinsonism (hemi-PD) can be induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB), disrupting dopaminergic neurons of the substantia nigra pars compacta (SNpc). In a previous study, we showed that subsequent injection of botulinum neurotoxin-A (BoNT-A) into the striatum can reverse most of the pathological motor symptoms and normalize the D2/D3R availability. To determine whether this rat model is suitable to explain olfactory deficits that occur in humans with PD, we examined the availability of D2/D3R by longitudinal [18F]fallypride-PET/CT, the density of tyrosine hydroxylase immunoreactivity in the OB, olfactory per...
Obesity is one of the most challenging diseases of the 21st century and is accompanied by behavio... more Obesity is one of the most challenging diseases of the 21st century and is accompanied by behavioural disorders. Exercise, dietary adjustments, or time-restricted feeding are the only successful long-term treatments to date. Fibroblast growth factor 21 (FGF21) plays a key role in dietary regulation, but FGF21 resistance is prevalent in obesity. The aim of this study was to investigate in obese mice whether weight reduction leads to improved behaviour and whether these behavioural changes are associated with decreased plasma FGF21 levels. After establishing a model for diet-induced obesity, mice were subjected to three different interventions for weight reduction, namely dietary change, treadmill exercise, or time-restricted feeding. In this study, we demonstrated that only the combination of dietary change and treadmill exercise affected all parameters leading to a reduction in weight, fat, and FGF21, as well as less anxious behaviour, higher overall activity, and improved olfactory...
Parkinson’s patients often suffer from depression and anxiety, for which there are no optimal tre... more Parkinson’s patients often suffer from depression and anxiety, for which there are no optimal treatments. Hemiparkinsonian (hemi-PD) rats were used to test whether intrastriatal Botulinum neurotoxin-A (BoNT-A) application could also have antidepressant-like properties in addition to the known improvement of motor performance. To quantify depression- and anxiety-like behavior, the forced swim test, tail suspension test, open field test, and elevated plus maze test were applied to hemi-PD rats injected with BoNT-A or vehicle. Furthermore, we correlated the results in the forced swim test, open field test, and elevated plus maze test with the rotational behavior induced by apomorphine and amphetamine. Hemi-PD rats did not show significant anxiety-like behavior as compared with Sham 6-OHDA- + Sham BoNT-A-injected as well as with non-injected rats. However, hemi-PD rats demonstrated increased depression-like behaviors compared with Sham- or non-injected rats; this was seen by increased s...
Introduction: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders... more Introduction: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders that has also been documented in Niemann-Pick disease type C1 (NPC1). NPC1 is a very rare, neurovisceral lipid storage disorder, characterized by a deficiency of Npc1 gene function that leads to progressive neurodegeneration. Here, we compared the pathologic effect of defective Npc1 gene on the vomeronasal neuroepithelium (VNE) with that of the olfactory epithelium (OE) in an NPC1 mouse model. Methods: Proliferation in the VNE and OE was assessed by applying a bromodeoxyuridine (BrdU) protocol. We further compared the immunoreactivities of anti-olfactory marker protein (OMP), and the lysosomal marker cathepsin-D in both epithelia. To investigate if degenerative effects of both olfactory systems can be prevented or reversed, some animals were treated with a combination of miglustat/allopregnanolone/2-hydroxypropyl-cyclodextrin (HPβCD), or a monotherapy with HPβCD alone. Results: Using Br...
Neurodegenerative diseases are often accompanied by olfactory deficits. Here we use a rare neurov... more Neurodegenerative diseases are often accompanied by olfactory deficits. Here we use a rare neurovisceral lipid storage disorder, Niemann-Pick disease C1 (NPC1), to illustrate disease-specific dynamics of olfactory dysfunction and its reaction upon therapy. Previous findings in a transgenic mouse model ( showed severe morphological and electrophysiological alterations of the olfactory epithelium (OE) and the olfactory bulb (OB) that ameliorated under therapy with combined 2-hydroxypropyl-ß-cyclodextrin (HPßCD)/allopregnanolone/miglustat or HPßCD alone. A buried pellet test was conducted to assess olfactory performance. qPCR for olfactory key markers and several olfactory receptors was applied to determine if their expression was changed under treatment conditions. In order to investigate the cell dynamics of the OB, we determined proliferative and apoptotic activities using a bromodeoxyuridine (BrdU) protocol and caspase-3 (cas-3) activity. Further, we performed immunohistochemistry ...
International journal of molecular sciences, Jan 24, 2018
Niemann-Pick-disease type C1 (NPC1) is an autosomal-recessive cholesterol-storage disorder. Besid... more 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-β-cyclodextrin (HPβCD), miglustat and allopregnanolone (combination therapy) with the previously established monotherapy using HPβCD. We examined transgene effects as well as treatment effects on liver morphology and hepatic lipid metabolism, focusing on hepatic cholesterol transporter genes. Livers ofmice showed hepatic cholesterol sequestration with consecutive liver injury, an increase of lipogenetic gene expression, e.g.,, a decrease of lipolytic gene expression, e.g.,and, and a decrease of lipid transporter gene expression, e.g.,,and. Both, combination therapy and ...
Background: Niemann-Pick type C1 (NPC1) disease is an inherited lysosomal storage disease caused ... more Background: Niemann-Pick type C1 (NPC1) disease is an inherited lysosomal storage disease caused by mutation of the Npc1 gene, resulting in a progressive accumulation of unesterified cholesterol and glycolipids in lysosomes of multiple tissues and leading to neurodegeneration and other disease. In Npc1 mutant mice, retinal degeneration including impaired visual function, lipofuscin accumulation in the pigment epithelium and ganglion cells as well as photoreceptor defects has been found. However, the pathologies of other individual cell types of the retina in Npc1 mutant mice are still not fully clear. We hypothesized that horizontal cells, amacrine cells, bipolar cells and glial cells are also affected in the retina of Npc1 mutant mice. Results: Immunohistochemistry and electron microscopy were used to investigate pathologies of ganglion cells, horizontal cells, amacrine cells, bipolar cells, and optic nerves as well as altered activity of glial cells in Npc1 mutant mice. Electron microscopy reveals that electron-dense inclusions are generally accumulated in ganglion cells, bipolar cells, Müller cells, and in the optic nerve. Furthermore, abnormal arborisation and ectopic processes of horizontal and amacrine cells as well as defective bipolar cells are observed by immunohistochemistry for specific cellular markers. Furthermore, hyperactivity of glial cells, including astrocytes, microglial cells, and Müller cells, is also revealed. Conclusions: Our data extend previous findings to show multiple defects in the retina of Npc1 mutant mice, suggesting an important role of Npc1 protein in the normal function of the retina.
Background: Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in th... more 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 2/2 ) 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 2/2 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 2/2 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 2/2 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.
Background: Niemann-Pick type C disease (NPC) is a rare autosomal recessive lipid storage disease... more Background: Niemann-Pick type C disease (NPC) is a rare autosomal recessive lipid storage disease characterized by progressive neurodegeneration. As only a few studies have been conducted on the impact of NPC on sensory systems, we used a mutant mouse model (NPC1 2/2 ) to examine the effects of this disorder to morphologically distinct regions of the olfactory system, namely the olfactory epithelium (OE) and olfactory bulb (OB). Methodology/Principal findings: For structural and functional analysis immunohistochemistry, electron microscopy, western blotting, and electrophysiology have been applied. For histochemistry and western blotting, we used antibodies against a series of neuronal and glia marker proteins, as well as macrophage markers. NPC1 2/2 animals present myelinlike lysosomal deposits in virtually all types of cells of the peripheral and central olfactory system. Especially supporting cells of the OE and central glia cells are affected, resulting in pronounced astrocytosis and microgliosis in the OB and other olfactory cortices. Up-regulation of Galectin-3, Cathepsin D and GFAP in the cortical layers of the OB underlines the critical role and location of the OB as a possible entrance gate for noxious substances. Unmyelinated olfactory afferents of the lamina propria seem less affected than ensheathing cells. Supporting the structural findings, electro-olfactometry of the olfactory mucosa suggests that NPC1 2/2 animals exhibit olfactory and trigeminal deficits. Conclusions/Significance: Our data demonstrate a pronounced neurodegeneration and glia activation in the olfactory system of NPC1 2/2 , which is accompanied by sensory deficits.
Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation ... more Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy. NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data. The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy. At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy. CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.
Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of... more Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of dopaminergic and cholinergic circuits. In Parkinson's disease (PD) imbalanced cholinergic hyperactivity prevails in the striatum. Interruption of acetylcholine (ACh) release in the striatum by locally injected botulinum neurotoxin A (BoNT-A) has been studied in the rat 6-hydroxydopamine (6-OHDA) model of PD (hemi-PD). The hemi-PD was induced by injection of 6-OHDA into the right medial forebrain bundle. Motor dysfunction provoked by apomorphine-induced contralateral rotation was completely reversed for more than 3 months by ipsilateral intrastriatal application of 1-2 ng BoNT-A. Interestingly, BoNT-A injected alone into the right striatum of naïve rats caused a slight transient ipsilateral apomorphine-induced rotation, which lasted only for about one month. Immunohistochemically, large axonal swellings appeared within the striatum injected with BoNT-A, which we tentatively named BoNT-A-induced varicosities. They contained either choline acetyltransferase or tyrosine hydroxylase. These findings suggest a selective inhibition of evoked release of ACh by locally applied BoNT-A. Intrastriatal application of BoNT-A may antagonize localized relative functional disinhibited hypercholinergic activity in neurodegenerative diseases such as PD avoiding side effects of systemic anti-cholinergic treatment.
Parkinson's disease (PD) is a neurodegenerative disorder involving several neuronal systems. Impa... more Parkinson's disease (PD) is a neurodegenerative disorder involving several neuronal systems. Impaired olfactory function may constitute one of the earliest symptoms of PD. However, it is still unclear to what degree changes of the olfactory epithelium may contribute to dysosmia and if these changes are different from those of other hyposmic or anosmic patients. This study aimed to investigate the hypothesis that olfactory loss in PD is a consequence of specific PDrelated damage of olfactory epithelium. Biopsies of 7 patients diagnosed with PD were taken. Six patients with PD were hyposmic, one anosmic. As non-PD controls served 9 patients with hyposmia, 9 with anosmia, and 7 normosmic individuals. Further, nasal mucosa of 4 postmortem individuals was investigated. Immunohistochemical examinations were performed with antibodies against olfactory marker protein (OMP), protein gene product 9.5 (PGP 9.5), beta-tubulin, (BT), proliferation associated antigen (Ki 67), the stem cell marker nestin, cytokeratin, p75NGFr, and a-synuclein. Most of the biopsy specimens exhibited irregular areas of olfactory-like, dysplastic epithelium positive for either PGP 9.5 or BT, but negative for OMP. No major histochemical differences in either the expression or distribution of these proteins were observed in the olfactory epithelium of patients with PD compared with controls. Reverse transcription PCR (RT-PCR) data indicated mRNA for OMP in almost all subjects, independently of their olfactory performance. These data support the idea that olfactory loss in Parkinson's disease is not a consequence of damage to the olfactory epithelium but rather results from distinct central-nervous abnormalities.
In contrast to many lower vertebrates, the vomeronasal epithelium (VNE) in humans has long been r... more In contrast to many lower vertebrates, the vomeronasal epithelium (VNE) in humans has long been regarded as absent or functionally irrelevant. For example, the neural connection between the VNE and the accessory olfactory bulb has been reported to degenerate during the second half of pregnancy and its presence has not been demonstrated in adults. Further, reports on the organ's occurrence in adult humans have been contradictory. The aims of this study were to collect immunohistochemical data on the neurogenic or epithelial character of the VNE [for example, with antibodies against protein gene product 9.5 (PGP 9.5), olfactory marker protein (OMP), beta-tubulin, and cytokeratin], determine its proliferative capacity (for example, proliferating cell nuclear antigen), as well as to examine the differentiation activity of VNE cells and their interactions with extracellular matrix components (for example, hyaluronan receptor CD44, galectins, and caveolin). To this end, we studied the vomeronasal organs (VNOs) of 22 human cadavers, three adult biopsies, one embryo (week 8) and one fetus (week 13) by means of immunohistochemistry. The histology of the VNE appeared extremely heterogeneous. There were sections of stratified, respiratory, and typical "pseudostratified" vomeronasal epithelia consisting of slender bipolar cells. Mostly negative immunohistochemical results for OMP indicated that the human VNE does not function like the mature olfactory epithelium. In addition, the investigations did not support the hypothesis that neural connections between the VNE and central brain structures might be present. On the other hand, the presence of some bipolar cells positive for both PGP 9.5 and soybean lectin (SBA) pointed to a neuron-like activity of a small subset of VNE cells. Proliferation antigens located in the nuclei of basally located cells of the VNE were not regularly expressed. However, positive reactions for CD44 demonstrated a high activity of VNE cells in terms of differentiation and migration. Some bipolar cells showed immunoreactivity for caveolin indicating its possible role in signal transduction and differentiation. In summary, the reaction patterns of most antibodies in the adult human VNE are different from those obtained in the olfactory epithelium and the VNO of the rat. However, the VNE shows a specific pattern of activity unique to the mucosa of the nasal cavity. Considering the histologically well differentiated epithelium and its steady maintenance, the VNE of the adult human appears to be a highly differentiated structure the function of which remains unclear.
The use of botulinum neurotoxins (BoNTs) for therapeutic purposes in neuromuscular disorders and ... more The use of botulinum neurotoxins (BoNTs) for therapeutic purposes in neuromuscular disorders and peripheral hypercholinergic conditions as well as in aesthetic medicine is widespread and common. BoNTs are also able to block the release of a wide range of transmitters from presynaptic boutons. Therefore, application of BoNTs directly in the central nervous system (CNS) is currently under study with respect to basic research and potentially as a new therapeutic strategy of neurological diseases. Investigations concentrate on effects of intracerebral and intraspinal application of BoNTs in rodents on the impact on spinal, nuclear, limbic and cortical neuronal circuits. In animal model first promising BoNT-induced therapeutical benefit has been shown in the treatment of pain, epilepsy, stroke and Parkinson's disease.
Niemann-Pick disease type C1 (NPC1) is a genetic neurovisceral disorder characterized by abnormal... more Niemann-Pick disease type C1 (NPC1) is a genetic neurovisceral disorder characterized by abnormalities in intracellular sterol trafficking. A knockout mouse model (NPC1) is an important tool for the study of pathogenesis and treatment strategies. In the present study, NPC1 mice were examined for pathological changes in the cornea. Fifteen inbred homozygous NPC1 knockout mice (NPC1, 5-10 weeks old), 5 age-matched heterozygous mice (NPC1), and 14 wild-type control mice (NPC1) were examined. In vivo confocal laser scanning microscopy (CLSM) was performed on both eyes of each animal; afterward, the eyes were processed for histology, electron microscopy, and lipid analysis. In vivo CLSM disclosed hyperreflective intracellular deposits in the intermediate and basal cell layers of corneal epithelium in all NPC1 mice. At the electron microscopy level, however, vacuolated cytoplasmic structures, 200-500 nm in diameter, with electron-dense material appeared in all structures investigated, including all epithelial layers and stromal keratocytes. These deposits were negative for filipin, a marker for unesterified cholesterol. Lipid analysis showed a marked increase in disialotetrahexosylganglioside 2 (GM2) level in NPC1 mice corneas, whereas no changes were detected in free cholesterol and disialotetrahexosylganglioside 3 (GM3) levels when compared with controls. Morphological changes characteristic for the NPC1 mouse cornea were visualized in all epithelial layers and keratocytes. In vivo CLSM findings were confirmed by other techniques. In vivo detection of ocular manifestations and analysis of ocular tissue have the potential to aid the diagnosis of NPC1 disease and to monitor the efficacy of treatment.
International journal of molecular sciences, Jan 6, 2017
Niemann-Pick disease type C1 (NPC1) is a fatal neurovisceral lysosomal lipid storage disorder. Th... more Niemann-Pick disease type C1 (NPC1) is a fatal neurovisceral lysosomal lipid storage disorder. The mutation of the NPC1 protein affects the homeostasis and transport of cholesterol and glycosphingolipids from late endosomes/lysosomes to the endoplasmic reticulum resulting in progressive neurodegeneration. Since olfactory impairment is one of the earliest symptoms in many neurodegenerative disorders, we focused on alterations of the olfactory epithelium in an NPC1 mouse model. Previous findings revealed severe morphological and immunohistochemical alterations in the olfactory system of NPC1(-/-) mutant mice compared with healthy controls (NPC1(+/+)). Based on immunohistochemical evaluation of the olfactory epithelium, we analyzed the impact of neurodegeneration in the olfactory epithelium of NPC1(-/-) mice and observed considerable loss of mature olfactory receptor neurons as well as an increased number of proliferating and apoptotic cells. Additionally, after administration of two d...
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-stan... more Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-standard method for analyzing modifications in gene expression in cells and tissues. However, large quantities of high-quality RNA samples are needed for analyzing the expression of multiple genes from one human tissue sample. Here, we provide an optimized protocol for extracting large amounts of RNA from human nasal mucosal biopsies. The quality and quantity of samples were sufficient for qRT-PCR analyses of the expressions of various genes, in duplicate. In contrast to other proto
Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is ... more Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD anima...
Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson’s disease (PD) in hu... more Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson’s disease (PD) in humans. The first central relay of the olfactory pathway, the olfactory bulb (OB), depends, among other things, on an intact, functional crosstalk between dopaminergic interneurons and dopamine receptors (D2/D3R). In rats, hemiparkinsonism (hemi-PD) can be induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB), disrupting dopaminergic neurons of the substantia nigra pars compacta (SNpc). In a previous study, we showed that subsequent injection of botulinum neurotoxin-A (BoNT-A) into the striatum can reverse most of the pathological motor symptoms and normalize the D2/D3R availability. To determine whether this rat model is suitable to explain olfactory deficits that occur in humans with PD, we examined the availability of D2/D3R by longitudinal [18F]fallypride-PET/CT, the density of tyrosine hydroxylase immunoreactivity in the OB, olfactory per...
Obesity is one of the most challenging diseases of the 21st century and is accompanied by behavio... more Obesity is one of the most challenging diseases of the 21st century and is accompanied by behavioural disorders. Exercise, dietary adjustments, or time-restricted feeding are the only successful long-term treatments to date. Fibroblast growth factor 21 (FGF21) plays a key role in dietary regulation, but FGF21 resistance is prevalent in obesity. The aim of this study was to investigate in obese mice whether weight reduction leads to improved behaviour and whether these behavioural changes are associated with decreased plasma FGF21 levels. After establishing a model for diet-induced obesity, mice were subjected to three different interventions for weight reduction, namely dietary change, treadmill exercise, or time-restricted feeding. In this study, we demonstrated that only the combination of dietary change and treadmill exercise affected all parameters leading to a reduction in weight, fat, and FGF21, as well as less anxious behaviour, higher overall activity, and improved olfactory...
Parkinson’s patients often suffer from depression and anxiety, for which there are no optimal tre... more Parkinson’s patients often suffer from depression and anxiety, for which there are no optimal treatments. Hemiparkinsonian (hemi-PD) rats were used to test whether intrastriatal Botulinum neurotoxin-A (BoNT-A) application could also have antidepressant-like properties in addition to the known improvement of motor performance. To quantify depression- and anxiety-like behavior, the forced swim test, tail suspension test, open field test, and elevated plus maze test were applied to hemi-PD rats injected with BoNT-A or vehicle. Furthermore, we correlated the results in the forced swim test, open field test, and elevated plus maze test with the rotational behavior induced by apomorphine and amphetamine. Hemi-PD rats did not show significant anxiety-like behavior as compared with Sham 6-OHDA- + Sham BoNT-A-injected as well as with non-injected rats. However, hemi-PD rats demonstrated increased depression-like behaviors compared with Sham- or non-injected rats; this was seen by increased s...
Introduction: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders... more Introduction: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders that has also been documented in Niemann-Pick disease type C1 (NPC1). NPC1 is a very rare, neurovisceral lipid storage disorder, characterized by a deficiency of Npc1 gene function that leads to progressive neurodegeneration. Here, we compared the pathologic effect of defective Npc1 gene on the vomeronasal neuroepithelium (VNE) with that of the olfactory epithelium (OE) in an NPC1 mouse model. Methods: Proliferation in the VNE and OE was assessed by applying a bromodeoxyuridine (BrdU) protocol. We further compared the immunoreactivities of anti-olfactory marker protein (OMP), and the lysosomal marker cathepsin-D in both epithelia. To investigate if degenerative effects of both olfactory systems can be prevented or reversed, some animals were treated with a combination of miglustat/allopregnanolone/2-hydroxypropyl-cyclodextrin (HPβCD), or a monotherapy with HPβCD alone. Results: Using Br...
Neurodegenerative diseases are often accompanied by olfactory deficits. Here we use a rare neurov... more Neurodegenerative diseases are often accompanied by olfactory deficits. Here we use a rare neurovisceral lipid storage disorder, Niemann-Pick disease C1 (NPC1), to illustrate disease-specific dynamics of olfactory dysfunction and its reaction upon therapy. Previous findings in a transgenic mouse model ( showed severe morphological and electrophysiological alterations of the olfactory epithelium (OE) and the olfactory bulb (OB) that ameliorated under therapy with combined 2-hydroxypropyl-ß-cyclodextrin (HPßCD)/allopregnanolone/miglustat or HPßCD alone. A buried pellet test was conducted to assess olfactory performance. qPCR for olfactory key markers and several olfactory receptors was applied to determine if their expression was changed under treatment conditions. In order to investigate the cell dynamics of the OB, we determined proliferative and apoptotic activities using a bromodeoxyuridine (BrdU) protocol and caspase-3 (cas-3) activity. Further, we performed immunohistochemistry ...
International journal of molecular sciences, Jan 24, 2018
Niemann-Pick-disease type C1 (NPC1) is an autosomal-recessive cholesterol-storage disorder. Besid... more 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-β-cyclodextrin (HPβCD), miglustat and allopregnanolone (combination therapy) with the previously established monotherapy using HPβCD. We examined transgene effects as well as treatment effects on liver morphology and hepatic lipid metabolism, focusing on hepatic cholesterol transporter genes. Livers ofmice showed hepatic cholesterol sequestration with consecutive liver injury, an increase of lipogenetic gene expression, e.g.,, a decrease of lipolytic gene expression, e.g.,and, and a decrease of lipid transporter gene expression, e.g.,,and. Both, combination therapy and ...
Background: Niemann-Pick type C1 (NPC1) disease is an inherited lysosomal storage disease caused ... more Background: Niemann-Pick type C1 (NPC1) disease is an inherited lysosomal storage disease caused by mutation of the Npc1 gene, resulting in a progressive accumulation of unesterified cholesterol and glycolipids in lysosomes of multiple tissues and leading to neurodegeneration and other disease. In Npc1 mutant mice, retinal degeneration including impaired visual function, lipofuscin accumulation in the pigment epithelium and ganglion cells as well as photoreceptor defects has been found. However, the pathologies of other individual cell types of the retina in Npc1 mutant mice are still not fully clear. We hypothesized that horizontal cells, amacrine cells, bipolar cells and glial cells are also affected in the retina of Npc1 mutant mice. Results: Immunohistochemistry and electron microscopy were used to investigate pathologies of ganglion cells, horizontal cells, amacrine cells, bipolar cells, and optic nerves as well as altered activity of glial cells in Npc1 mutant mice. Electron microscopy reveals that electron-dense inclusions are generally accumulated in ganglion cells, bipolar cells, Müller cells, and in the optic nerve. Furthermore, abnormal arborisation and ectopic processes of horizontal and amacrine cells as well as defective bipolar cells are observed by immunohistochemistry for specific cellular markers. Furthermore, hyperactivity of glial cells, including astrocytes, microglial cells, and Müller cells, is also revealed. Conclusions: Our data extend previous findings to show multiple defects in the retina of Npc1 mutant mice, suggesting an important role of Npc1 protein in the normal function of the retina.
Background: Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in th... more 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 2/2 ) 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 2/2 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 2/2 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 2/2 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.
Background: Niemann-Pick type C disease (NPC) is a rare autosomal recessive lipid storage disease... more Background: Niemann-Pick type C disease (NPC) is a rare autosomal recessive lipid storage disease characterized by progressive neurodegeneration. As only a few studies have been conducted on the impact of NPC on sensory systems, we used a mutant mouse model (NPC1 2/2 ) to examine the effects of this disorder to morphologically distinct regions of the olfactory system, namely the olfactory epithelium (OE) and olfactory bulb (OB). Methodology/Principal findings: For structural and functional analysis immunohistochemistry, electron microscopy, western blotting, and electrophysiology have been applied. For histochemistry and western blotting, we used antibodies against a series of neuronal and glia marker proteins, as well as macrophage markers. NPC1 2/2 animals present myelinlike lysosomal deposits in virtually all types of cells of the peripheral and central olfactory system. Especially supporting cells of the OE and central glia cells are affected, resulting in pronounced astrocytosis and microgliosis in the OB and other olfactory cortices. Up-regulation of Galectin-3, Cathepsin D and GFAP in the cortical layers of the OB underlines the critical role and location of the OB as a possible entrance gate for noxious substances. Unmyelinated olfactory afferents of the lamina propria seem less affected than ensheathing cells. Supporting the structural findings, electro-olfactometry of the olfactory mucosa suggests that NPC1 2/2 animals exhibit olfactory and trigeminal deficits. Conclusions/Significance: Our data demonstrate a pronounced neurodegeneration and glia activation in the olfactory system of NPC1 2/2 , which is accompanied by sensory deficits.
Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation ... more Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy. NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data. The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy. At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy. CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.
Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of... more Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of dopaminergic and cholinergic circuits. In Parkinson's disease (PD) imbalanced cholinergic hyperactivity prevails in the striatum. Interruption of acetylcholine (ACh) release in the striatum by locally injected botulinum neurotoxin A (BoNT-A) has been studied in the rat 6-hydroxydopamine (6-OHDA) model of PD (hemi-PD). The hemi-PD was induced by injection of 6-OHDA into the right medial forebrain bundle. Motor dysfunction provoked by apomorphine-induced contralateral rotation was completely reversed for more than 3 months by ipsilateral intrastriatal application of 1-2 ng BoNT-A. Interestingly, BoNT-A injected alone into the right striatum of naïve rats caused a slight transient ipsilateral apomorphine-induced rotation, which lasted only for about one month. Immunohistochemically, large axonal swellings appeared within the striatum injected with BoNT-A, which we tentatively named BoNT-A-induced varicosities. They contained either choline acetyltransferase or tyrosine hydroxylase. These findings suggest a selective inhibition of evoked release of ACh by locally applied BoNT-A. Intrastriatal application of BoNT-A may antagonize localized relative functional disinhibited hypercholinergic activity in neurodegenerative diseases such as PD avoiding side effects of systemic anti-cholinergic treatment.
Parkinson's disease (PD) is a neurodegenerative disorder involving several neuronal systems. Impa... more Parkinson's disease (PD) is a neurodegenerative disorder involving several neuronal systems. Impaired olfactory function may constitute one of the earliest symptoms of PD. However, it is still unclear to what degree changes of the olfactory epithelium may contribute to dysosmia and if these changes are different from those of other hyposmic or anosmic patients. This study aimed to investigate the hypothesis that olfactory loss in PD is a consequence of specific PDrelated damage of olfactory epithelium. Biopsies of 7 patients diagnosed with PD were taken. Six patients with PD were hyposmic, one anosmic. As non-PD controls served 9 patients with hyposmia, 9 with anosmia, and 7 normosmic individuals. Further, nasal mucosa of 4 postmortem individuals was investigated. Immunohistochemical examinations were performed with antibodies against olfactory marker protein (OMP), protein gene product 9.5 (PGP 9.5), beta-tubulin, (BT), proliferation associated antigen (Ki 67), the stem cell marker nestin, cytokeratin, p75NGFr, and a-synuclein. Most of the biopsy specimens exhibited irregular areas of olfactory-like, dysplastic epithelium positive for either PGP 9.5 or BT, but negative for OMP. No major histochemical differences in either the expression or distribution of these proteins were observed in the olfactory epithelium of patients with PD compared with controls. Reverse transcription PCR (RT-PCR) data indicated mRNA for OMP in almost all subjects, independently of their olfactory performance. These data support the idea that olfactory loss in Parkinson's disease is not a consequence of damage to the olfactory epithelium but rather results from distinct central-nervous abnormalities.
In contrast to many lower vertebrates, the vomeronasal epithelium (VNE) in humans has long been r... more In contrast to many lower vertebrates, the vomeronasal epithelium (VNE) in humans has long been regarded as absent or functionally irrelevant. For example, the neural connection between the VNE and the accessory olfactory bulb has been reported to degenerate during the second half of pregnancy and its presence has not been demonstrated in adults. Further, reports on the organ's occurrence in adult humans have been contradictory. The aims of this study were to collect immunohistochemical data on the neurogenic or epithelial character of the VNE [for example, with antibodies against protein gene product 9.5 (PGP 9.5), olfactory marker protein (OMP), beta-tubulin, and cytokeratin], determine its proliferative capacity (for example, proliferating cell nuclear antigen), as well as to examine the differentiation activity of VNE cells and their interactions with extracellular matrix components (for example, hyaluronan receptor CD44, galectins, and caveolin). To this end, we studied the vomeronasal organs (VNOs) of 22 human cadavers, three adult biopsies, one embryo (week 8) and one fetus (week 13) by means of immunohistochemistry. The histology of the VNE appeared extremely heterogeneous. There were sections of stratified, respiratory, and typical "pseudostratified" vomeronasal epithelia consisting of slender bipolar cells. Mostly negative immunohistochemical results for OMP indicated that the human VNE does not function like the mature olfactory epithelium. In addition, the investigations did not support the hypothesis that neural connections between the VNE and central brain structures might be present. On the other hand, the presence of some bipolar cells positive for both PGP 9.5 and soybean lectin (SBA) pointed to a neuron-like activity of a small subset of VNE cells. Proliferation antigens located in the nuclei of basally located cells of the VNE were not regularly expressed. However, positive reactions for CD44 demonstrated a high activity of VNE cells in terms of differentiation and migration. Some bipolar cells showed immunoreactivity for caveolin indicating its possible role in signal transduction and differentiation. In summary, the reaction patterns of most antibodies in the adult human VNE are different from those obtained in the olfactory epithelium and the VNO of the rat. However, the VNE shows a specific pattern of activity unique to the mucosa of the nasal cavity. Considering the histologically well differentiated epithelium and its steady maintenance, the VNE of the adult human appears to be a highly differentiated structure the function of which remains unclear.
The use of botulinum neurotoxins (BoNTs) for therapeutic purposes in neuromuscular disorders and ... more The use of botulinum neurotoxins (BoNTs) for therapeutic purposes in neuromuscular disorders and peripheral hypercholinergic conditions as well as in aesthetic medicine is widespread and common. BoNTs are also able to block the release of a wide range of transmitters from presynaptic boutons. Therefore, application of BoNTs directly in the central nervous system (CNS) is currently under study with respect to basic research and potentially as a new therapeutic strategy of neurological diseases. Investigations concentrate on effects of intracerebral and intraspinal application of BoNTs in rodents on the impact on spinal, nuclear, limbic and cortical neuronal circuits. In animal model first promising BoNT-induced therapeutical benefit has been shown in the treatment of pain, epilepsy, stroke and Parkinson's disease.
Niemann-Pick disease type C1 (NPC1) is a genetic neurovisceral disorder characterized by abnormal... more Niemann-Pick disease type C1 (NPC1) is a genetic neurovisceral disorder characterized by abnormalities in intracellular sterol trafficking. A knockout mouse model (NPC1) is an important tool for the study of pathogenesis and treatment strategies. In the present study, NPC1 mice were examined for pathological changes in the cornea. Fifteen inbred homozygous NPC1 knockout mice (NPC1, 5-10 weeks old), 5 age-matched heterozygous mice (NPC1), and 14 wild-type control mice (NPC1) were examined. In vivo confocal laser scanning microscopy (CLSM) was performed on both eyes of each animal; afterward, the eyes were processed for histology, electron microscopy, and lipid analysis. In vivo CLSM disclosed hyperreflective intracellular deposits in the intermediate and basal cell layers of corneal epithelium in all NPC1 mice. At the electron microscopy level, however, vacuolated cytoplasmic structures, 200-500 nm in diameter, with electron-dense material appeared in all structures investigated, including all epithelial layers and stromal keratocytes. These deposits were negative for filipin, a marker for unesterified cholesterol. Lipid analysis showed a marked increase in disialotetrahexosylganglioside 2 (GM2) level in NPC1 mice corneas, whereas no changes were detected in free cholesterol and disialotetrahexosylganglioside 3 (GM3) levels when compared with controls. Morphological changes characteristic for the NPC1 mouse cornea were visualized in all epithelial layers and keratocytes. In vivo CLSM findings were confirmed by other techniques. In vivo detection of ocular manifestations and analysis of ocular tissue have the potential to aid the diagnosis of NPC1 disease and to monitor the efficacy of treatment.
International journal of molecular sciences, Jan 6, 2017
Niemann-Pick disease type C1 (NPC1) is a fatal neurovisceral lysosomal lipid storage disorder. Th... more Niemann-Pick disease type C1 (NPC1) is a fatal neurovisceral lysosomal lipid storage disorder. The mutation of the NPC1 protein affects the homeostasis and transport of cholesterol and glycosphingolipids from late endosomes/lysosomes to the endoplasmic reticulum resulting in progressive neurodegeneration. Since olfactory impairment is one of the earliest symptoms in many neurodegenerative disorders, we focused on alterations of the olfactory epithelium in an NPC1 mouse model. Previous findings revealed severe morphological and immunohistochemical alterations in the olfactory system of NPC1(-/-) mutant mice compared with healthy controls (NPC1(+/+)). Based on immunohistochemical evaluation of the olfactory epithelium, we analyzed the impact of neurodegeneration in the olfactory epithelium of NPC1(-/-) mice and observed considerable loss of mature olfactory receptor neurons as well as an increased number of proliferating and apoptotic cells. Additionally, after administration of two d...
1 Allgemeine Zellbiologie, Zellteilung und Zelltod – 2
2 Genetik – 16
3 Grundlagen der Mikrobio... more 1 Allgemeine Zellbiologie, Zellteilung und Zelltod – 2 2 Genetik – 16 3 Grundlagen der Mikrobiologie und Ökologie – 28
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Papers by Martin Witt
2 Genetik – 16
3 Grundlagen der Mikrobiologie und Ökologie – 28