Genetic dissection of strain dependent paraquat-induced neurodegeneration in the substantia nigra pars compacta

The etiology of the vast majority of Parkinson's disease (PD) cases is unknown. It is generally accepted that there is an interaction between exposures to environmental agents with underlying genetic sensitivity. Recent epidemiological studies have shown that people living in agricultural communities have an increased risk of PD. Within these communities, paraquat (PQ) is one of the most utilized herbicides. PQ acts as a direct redox cycling agent to induce formation of free radicals and when administered to mice induces the cardinal symptoms of parkinsonism, including loss of TH+-positive dopaminergic (DA) neurons in the ventral midbrain's substantia nigra pars compacta (SNpc). Here we show that PQ-induced SNpc neuron loss is highly dependent on genetic background: C57BL/6J mice rapidly lose ～50% of their SNpc DA neurons, whereas inbred Swiss-Webster (SWR/J) mice do not show any significant loss. We intercrossed these two strains to map quantitative trait loci (QTLs) that underlie PQ-induced SNpc neuron loss. Using genome-wide linkage analysis we detected two significant QTLs. The first is located on chromosome 5 (Chr 5) centered near D5Mit338, whereas the second is on Chr 14 centered near D14Mit206. These two QTLs map to different loci than a previously identified QTL (Mptp1) that controls a significant portion of strain sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), suggesting that the mechanism of action of these two parkinsonian neurotoxins are different.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1?μM tetrodotoxin decreased TH expression beginning around 20?h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300?nM apamin increased TH mRNA and protein between 12 and 24?h later in slices of adult midbrain. Two-week infusions of 300?nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ～20?h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.     

Zinc bone loss in chronic renal failure and chronic metabolic acidosis

The effects of chronic metabolic acidosis (CMA) on zinc (Zn) bone content and urinary excretion were examined in the presence of normal or reduced renal function together with some aspects of calcium (Ca) metabolism. Four groups of rats were compared. All were fed a 30% protein and 9 mg Zn/100 g diet. Two were uremic (U): The first developed acidosis (UA), which was suppressed in the other (UNA) by NaHCO₃ supplement. Two other groups had normal renal function: One was normal (CNA), and the other had NH₄Cl in the drinking water and acidosis (CA). Femur total Zn and Ca content was markedly reduced by CMA and was not affected by uremia. Zn urinary excretion was increased by CMA and unaltered by uremia. Ca urinary excretion was markedly reduced in uremic rats, but was enhanced in both acidotic conditions. Urinary Ca and Zn showed a strong correlation in uremic and in control rats. Plasma parathormone and 1,25(OH)₂D₃ were unchanged by CMA. These data are in agreement with a direct primary effect of CMA on bone in releasing buffers. CMA induces bone resorption and a parallel decrease of mineral bone components, such as Ca and Zn, with little or no role of PTH, 1,25(OH)₂D₃ and of uremia itself.     

The OPG/RANKL/RANK system in metabolic bone diseases

The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. Various studies suggest that human metabolic bone diseases are related to alterations of this system. Here we summarize OPG/RANKL/RANK abnormalities in different forms of osteoporoses and hyperparathyroidism. Skeletal estrogen agonists (including 17beta-estradiol, raloxifene, and genistein) induce osteoblastic OPG production through estrogen receptor-alpha activation in vitro, while immune cells appear to over-express RANKL in estrogen deficiency in vivo. Of note, OPG administration can prevent bone loss associated with estrogen deficiency as observed in both animal models and a small clinical study. Glucocorticoids and immunosuppressants concurrently up-regulate RANKL and suppress OPG in osteoblastic cells in vitro, and glucocorticoids are among the most powerful drugs to suppress OPG serum levels in vivo. As for mechanisms of immobilization-induced bone loss, it appears that mechanical strain inhibits RANKL production through the ERK 1/2 MAP kinase pathway and up-regulates OPG production in vitro. Hence, lack of mechanical strainduring immobilization may favor an enhanced RANKL-to-OPG ratio leading to increased bone loss. As for hyperparathyroidism, chronic PTH exposure concurrently enhances RANKL production and suppresses OPG secretion through activation of osteoblastic protein kinase A in vitro which would favour increased osteoclastic activity. In sum, the capacity for OPG to antagonize the increases in bone loss seen in many rodent models of metabolic bone disease implicates RANKL/OPG imbalances as the likely etiology and supports the potential role for a RANKL antagonist as a therapeutic intervention in these settings.     

The development of denosumab for the treatment of diseases of bone loss and cancer-induced bone destruction

Denosumab is a fully human monoclonal antibody against RANK ligand (RANKL), an essential cytokine for the formation, function, and survival of osteoclasts. The role of excessive RANKL as a contributor to conditions characterized by bone loss or bone destruction has been well studied. With its novel mechanism of action, denosumab offers a significant advance in the treatment of postmenopausal osteoporosis; bone loss associated with hormone ablation therapy in women with breast cancer and men with prostate cancer; and the prevention of skeletal-related events in patients with bone metastases from solid tumors by offering clinical benefit to these patients in need.     

Distinctive diagnostic features of material aspirated from marrow in metabolic bone diseases

In a series of cases of Paget's disease of the bone, two types of cells not previously described were observed in material aspirated from bone marrow in areas of osteitis deformans. One type was mononuclear, the other was giant, multinucleated and syncytial. They have been identified as osteoblasts and osteoclasts, respectively. The identification was based mainly on correlation with the histologic picture of osteitis deformans and of normal-growing bones as seen in section studies. Both osteoblasts and osteoclasts were recovered in aspirated bone marrow material in other instances of metabolic bone diseases associated with increased bone repair and bone resorption-in hyperparathyroidism, osteoblastic malignant lesions, rickets, hemolytic anemia in children, and in normal infants in the growth zone of bone in the tibia. They were not seen in senile and postmenopausal osteoporosis. Recognition of osteoblasts and osteoclasts in smear preparations from aspirated bone marrow material may serve as a diagnostic aid in metabolic bone diseases. The differentiation of osteoblasts from neoplastic cells is important in cases in which metastatic cancer of the bone is suspected and x-ray findings are inconclusive.     

Developmental loss and resistance to MPTP toxicity of dopaminergic neurones in substantia nigra pars compacta of gamma-synuclein, alpha-synuclein and double alpha/gamma-synuclein null mutant mice

The growing body of evidence suggests that intermediate products of alpha-synuclein aggregation cause death of sensitive populations of neurones, particularly dopaminergic neurones, which is a critical event in the development of Parkinson's disease and other synucleinopathies. The role of two other members of the family, beta-synuclein and gamma-synuclein, in neurodegeneration is less understood. We studied the effect of inactivation of gamma-synuclein gene on mouse midbrain dopaminergic neurones. Reduced number of dopaminergic neurones was found in substantia nigra pars compacta (SNpc) but not in ventral tegmental area (VTA) of early post-natal and adult gamma-synuclein null mutant mice. Similar reductions were revealed in alpha-synuclein and double alpha-synuclein/gamma-synuclein null mutant animals. However, in none of these mutants did this lead to significant changes of striatal dopamine or dopamine metabolite levels and motor dysfunction. In all three studied types of null mutants, dopaminergic neurones of SNpc were resistant to methyl-phenyl-tetrahydropyridine (MPTP) toxicity. We propose that both synucleins are important for effective survival of SNpc neurones during critical period of development but, in the absence of these proteins, permanent activation of compensatory mechanisms allow many neurones to survive and become resistant to certain toxic insults.     

FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain

We have isolated cDNA encoding a novel FGF (212 amino acids) from rat brain. Because this is the 20th documented member of the FGF family, we tentatively term it FGF-20. Among FGF family members, FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62% amino acid identity, respectively). Human FGF-20 gene was found in the human genomic sequence mapped to the 8p21.3-p22 region. Human FGF-20 is highly identical to rat FGF-20 (95% amino acid identity). FGF-20 mRNA was preferentially expressed in rat brain among the adult major tissues examined. The localization of FGF-20 mRNA in rat brain was also examined by in situ hybridization. FGF-20 mRNA was preferentially expressed in the substantia nigra pars compacta. To examine the biological activity of FGF-20, recombinant rat FGF-20 was produced by insect cells infected with recombinant baculovirus containing rat FGF-20 cDNA. Recombinant rat FGF-20 enhanced the survival of midbrain dopaminergic neurons. The present results indicate that FGF-20 is a novel neurotrophic factor preferentially expressed in the substantia nigra pars compacta of rat brain.     

Voltage-operated Ca2+ channels regulate dopamine release from somata of dopamine neurons in the substantia nigra pars compacta

Dopamine (DA) neurons release DA not only from axon terminals at the striatum, but from their somata and dendrites at the substantia nigra pars compacta (SNc). Released DA may auto-regulate further DA release or modulate non-DA cells. However, the actual mechanism of somatodendritic DA release, especially the Ca²⁺ dependency of the process, remains controversial. In this study, we used amperometry to monitor DA release from somata of acutely isolated rat DA neurons. We found that DA neurons spontaneously released DA in the resting state. Removal of extracellular Ca²⁺ and application of blockers for voltage-operated Ca²⁺ channels (VOCCs) suppressed the frequency of secretion events. Activation of VOCCs by stimulation with K⁺-rich saline increased the frequency of secretion events, which were also sensitive to blockers for L- and T-type Ca²⁺ channels. These results suggest that Ca²⁺ influx through VOCCs regulates DA release from somata of DA neurons.     

The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area.

The dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area play a crucial role in regulating movement and cognition respectively. Several lines of evidence suggest that a degeneration of dopaminergic cells in the substantia nigra produces the symptoms of Parkinson's disease. On the other hand, a hyperactivity of the dopaminergic transmission in the brain induces dyskinesia, dystonia and psychosis. It is also well established that the euphoric and rewarding responses evoked by drugs of addiction, such as amphetamine and cocaine, are mediated by central dopamine systems. Electrophysiological experiments which study the activity of single dopaminergic neurons in the ventral mesencephalon have shown that dopamine and dopaminergic drugs reduce the firing frequency of these cells. This is due to the stimulation of D2-D3 autoreceptors and to a hyperpolarization of the membrane produced by an increase in potassium conductance. In addition, substances which increase the release (amphetamine), the synthesis (levodopa) or block the uptake (cocaine, nomifensine, amineptine) of dopamine in the brain inhibit the firing activity of the dopaminergic cells throughout dopamine-mediated mechanisms. In this review, we will briefly examine the literature concerning the physiological and behavioural responses caused by dopamine and dopaminergic agents on the dopaminergic neurons of the ventral mesencephalon. Our conclusion suggests that the electrophysiological actions of dopamine and dopamine-related drugs on dopaminergic cells in the ventral mesencephalon might be indicative of the pharmacological effects of these agents on the brain.     

Membrane-permeable Bcl-xL prevents MPTP-induced dopaminergic neuronal loss in the substantia nigra

The anti-apoptotic Bcl-x_L is a promising agent to prevent neurodegeneration in Parkinson's disease, which is characterized by a demise of dopaminergic neurons. We linked Bcl-x_L to a peptide that allows its delivery across biological membranes and the blood–brain barrier. We tested the fusion protein in two models of Parkinson's Disease. Cell-permeable Bcl-x_L protected neuroblastoma cells from the selective neurotoxin 1-methyl-4-phenylpyridinium. Furthermore, its systemic application in aged mice protected dopaminergic neurons following administration of MPTP as revealed by counting of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta . Hence, we present that a cell-permeable form of an anti-apoptotic protein can be delivered to CNS neurons through its systemic application, and we provide the proof that the delivery of this protein to the CNS neurons effectively prevents neuronal cell death in models of chronic neurodegenerative diseases.     

Antibodies in metabolic diseases

In the past century, incidences of chronic metabolic diseases, such as obesity and type II diabetes, have increased dramatically. Obesity and abnormal insulin level are associated with a wide variety of health problems including a markedly increased risk for type II diabetes, fatty liver, hepato-biliary and gallbladder diseases, cardiovascular pathologies, neurodegenerative disorders, asthma and a variety of cancers. The development of therapeutic antibodies has evolved over the past decades into a mainstay of therapeutic options for patients with inflammatory diseases and cancer, while other indication areas such as metabolic diseases have so far only been rarely addressed. Although therapeutic antibodies might have advantages over current type II diabetes treatments like favorable serum half-life and high specificity, their development is also likely to face obstacles. For example the technical feasibility of antibody generation against G protein coupled receptors and transporters is challenging, patient compliance for a likely needle application might be limited, bioavailability in organs involved in the pathogenesis like the brain might be suboptimal and reimbursement issues for high treatment costs have to be taken into account. The current review focuses on the pathogenesis and standard therapeutic approaches as well as antibodies in development and potential antibody targets for type II diabetes.     

Brief communication: on the optimum number of metacarpals for roentgenogrammetric measurement

Interpretation of dental development of fossil hominids requires understanding of and comparison with the pattern and timing of dental development among living humans and pongids. We report the first study of crown and root calcification in the lower permanent molar teeth among chimpanzees (Pan troglodytes) of known chronological age. A series of 99 lateral head radiographs of 16 captive-born chimpanzees were analyzed. Radiographs were taken at irregular intervals throughout the entire postnatal period of dental development from birth to 13 years of age. Permanent mandibular molars were rated on an eight-point maturation scale from initial radiographic appearance through crown and root calcification and apical closure of the root canals. In addition, we were able to document initial crown calcification and completion, as well as root completion and apical closure in incisors, canines, and premolars. Our results show several differences from the widely cited developmental schedule for pongid dentitions of Dean and Wood (Folia Primatol. 36:111–127, 1981). We found a much greater degree of temporal overlap in calcification of the crowns of adjacent molars, a pattern very unlike that usually seen in human dental development, which is characterized by delays between the onset of crown calcification in the molar series. Also, the ages and durations of crown and root formation in our chimp sample differ from the estimates proposed by Dean and Wood. By more clearly establishing the nature of developmental schedules and the timing of major events in the pongid dentition, these results should aid in the ongoing controversies concerning the human or pongid nature of dental development among Plio-Pleistocene hominids.     

The lesion of the rat substantia nigra pars compacta dopaminergic neurons as a model for Parkinson's disease memory disabilities

1. In this article we review the studies of memory disabilities in a rat model o Parkinson's disease (PD).2. Intranigral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to rats causes a partial lesion in the substantia nigra, compact part (SNc) and a specific loss of dopamine and its metabolites in the striatum of rats.3. These animals present learning and memory deficits but no sensorimotor impairments, thus modeling the early phase of PD when cognitive impairments are observed but the motor symptoms of the disease are barely present.4. The cognitive deficits observed in these animals affect memory tasks proposed to model habit learning (the cued version of the water maze task and the two-way active avoidance task) and working memory (a working memory version of the water maze), but spare long-term spatial memory (the spatial reference version of the Morris water maze).5. The treatment of these animals with levodopa in a dose that restores the striatal level of dopamine does not reverse these memory impairments, probably because this treatment promotes a high level of dopamine in extrastriatal brain regions, such as the prefrontal cortex and the hippocampus.6. On the other hand, the adenosine receptor antagonist, caffeine, partly reverse the memory impairment effect of SNc lesion in these rats. This effect may be due to caffeine action on nigrostriatal neurons, since it induces dopamine release and modulates the interaction between adenosine and dopamine receptor activity.7. These results suggest that the MPTP SNc-lesioned rats are a good model to study memory disabilities related to PD and that caffeine and other selective A(2A) adenosine receptor antagonists are promising drugs to treat this symptoms in PD patients.     

New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis

Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.