Olfactory Dysfunction and Neurotransmitter Disturbance in Olfactory Bulb of Transgenic Mice Expressing Human A53T Mutant α-Synuclein

Parkinson disease is a multi-system neurodegenerative disease characterized by both motor and non-motor symptoms. Hyposmia is one of the early non-motor symptoms occurring in more than 90% of Parkinson disease cases, which can precede motor symptoms even several years. Up to now, the relationship between hyposmia and Parkinson disease remains elusive. Lack of proper animal models of hyposmia restricts the investigation. In this study we assessed olfactory function in Prp-A53T-α-synuclein transgenic (αSyn^A53T) mice which had been reported to show age-dependent motor impairments and intracytoplasmic inclusions. We also examined cholinergic and dopaminergic systems in olfactory bulb of αSyn^A53T mice by immunofluorescent staining, enzyme linked immunosorbent assay and western blot. We found that compared to wild type littermates, αSyn^A53T mice at 6 months or older displayed a deficit of odor discrimination and odor detection. No significant changes were found in olfactory memory and odor habituation. Furthermore compared to wildtype littermates, in olfactory bulb of αSyn^A53T mice at 10 months old we detected a marked decrease of cholinergic neurons in mitral cell layer and a decrease of acetylcholinesterase activity, while dopaminergic neurons were found increased in glomerular layer, accompanied with an increase of tyrosine hydroxylase protein. Our studies indicate that αSyn^A53T mice have olfactory dysfunction before motor deficits occur, and the cholinergic and dopaminergic disturbance might be responsible for the Parkinson disease-related olfactory dysfunction.     

Brain β-Amyloid Accumulation in Transgenic Mice Expressing Mutant Superoxide Dismutase 1

Oxidative stress is implicated in both the deposition and pathogenesis of -amyloid (A) protein in Alzheimers disease (AD). Accordingly, overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) in neuronal cells and transgenic AD mice reduces A toxicity and accumulation. In contrast, mutations in SOD1 associated with amyotrophic lateral sclerosis (ALS) confer enhanced pro-oxidative enzyme activities. We therefore examined whether ALS-linked mutant SOD1 overexpression in motor neuronal cells or transgenic ALS mice modulates A toxicity or its accumulation in the brain. Aggregated, but not freshly solubilised, substrate-bound A peptides induced degenerative morphology and cytotoxicity in motor neuron-like NSC-34 cells. Transfection of NSC-34 cells with human wild-type SOD1 attenuated A-induced toxicity, however this neuroprotective effect was also observed for ALS-linked mutant SOD1. Analysis of the cerebral cortex, brainstem, cerebellum and olfactory bulb from transgenic SOD1^G93A mice using enzyme-linked immunosorbent assay of acid–guanidine extracts revealed age-dependent elevations in A levels, although not significantly different from wild-type mouse brain. In addition, brain amyloid protein precursor (APP) levels remained unaltered as a consequence of mutant SOD1 expression. We therefore conclude that mutant SOD1 overexpression promotes neither A toxicity nor brain accumulation in these ALS models.     

Longitudinal Metabolomics Profiling of Parkinson’s Disease-Related α-Synuclein A53T Transgenic Mice

Metabolic homeostasis is critical for all biological processes in the brain. The metabolites are considered the best indicators of cell states and their rapid fluxes are extremely sensitive to cellular changes. While there are a few studies on the metabolomics of Parkinson’s disease, it lacks longitudinal studies of the brain metabolic pathways affected by aging and the disease. Using ultra-high performance liquid chromatography and tandem mass spectroscopy (UPLC/MS), we generated the metabolomics profiling data from the brains of young and aged male PD-related α-synuclein A53T transgenic mice as well as the age- and gender-matched non-transgenic (nTg) controls. Principal component and unsupervised hierarchical clustering analyses identified distinctive metabolites influenced by aging and the A53T mutation. The following metabolite set enrichment classification revealed the alanine metabolism, redox and acetyl-CoA biosynthesis pathways were substantially disturbed in the aged mouse brains regardless of the genotypes, suggesting that aging plays a more prominent role in the alterations of brain metabolism. Further examination showed that the interaction effect of aging and genotype only disturbed the guanosine levels. The young A53T mice exhibited lower levels of guanosine compared to the age-matched nTg controls. The guanosine levels remained constant between the young and aged nTg mice, whereas the aged A53T mice showed substantially increased guanosine levels compared to the young mutant ones. In light of the neuroprotective function of guanosine, our findings suggest that the increase of guanosine metabolism in aged A53T mice likely represents a protective mechanism against neurodegeneration, while monitoring guanosine levels could be applicable to the early diagnosis of the disease.     

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated α-synuclein (αS^D) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T αS and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of αS^D (disease-associated αS) in these mice, an ELISA was designed to specifically quantify αS^D in sick mice. Analysis of the central nervous system in this mouse model showed the presence of αS^D mainly in the caudal brain regions and the spinal cord. There were no differences in αS^D distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of αS^D immunoreactivity using an antibody against Ser129 phosphorylated αS by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of αS. The propagation of αS^D, suggesting the involvement of a “prion-like” mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.     

Dopaminergic Toxin 1-Methyl-4-Phenylpyridinium,Proteins α-Synuclein and Glia Maturation Factor Activate Mast Cells and Release Inflammatory Mediators

Parkinson’s disease (PD) is characterized by the presence of Lewy bodies and degeneration of dopaminergic neurons. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Lewy body component α-synuclein activates glia in PD pathogenesis. Mast cells and glia maturation factor (GMF) are implicated in neuroinflammatory conditions including Multiple Sclerosis. However, the role of mast cells in PD is not yet known. We have analyzed the effect of recombinant GMF, MPP+, α-synuclein and interleukin-33 (IL-33) on mouse bone marrow-derived cultured mast cells (BMMCs), human umbilical cord blood-derived cultured mast cells (hCBMCs) and mouse brain-derived cultured astrocytes by quantifying cytokines/chemokines released using ELISA or by detecting the expression of co-stimulatory molecules CD40 and CD40L by flow cytometry. GMF significantly released chemokine (C-C motif) ligand 2 (CCL2) from BMMCs but its release was reduced in BMMCs from GMF knockout mice. GMF, α-synuclein and MPP+ released IL-1β, β-hexosaminidase from BMMCs, and IL-8 from hCBMCs. GMF released CCL5, and IL-33- induced the expression of GMF from hCBMCs. Novel GMF expression was detected in hCBMCs and BMMCs by immunocytochemistry. GMF released tumor necrosis factor-alpha (TNF-α) from mouse astrocytes, and this release was greater in BMMC- astrocyte coculture than in individual cultures. Flow cytometry results showed increased IL-33 expression by GMF and MPP+, and GMF-induced CD40 expression in astrocytes. Proinflammatory mediator release by GMF, MPP+ and α-synuclein, as well as GMF expression by mast cells indicate a potential therapeutic target for neurodegenerative diseases including PD.     

Mutant α-synuclein and aging reduce neurogenesis in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

Neurogenesis, the production of new neurons from less differentiated precursor cells, normally occurs in adult brains in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Neurogenesis declines with aging. In previous studies, neurogenesis was stimulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) in young animals. In this study, we examined the effect of acute MPTP administration and mutant α-synuclein A53T on neurogenesis and migration of newborn neurons in the aged (23-month) vs. young (2-month) rodent brain. Cell proliferation and neurogenesis were assessed via bromodeoxyuridine labeling and immunostaining for cell type-specific markers. In the aged brain, neural precursor cells in the rostral SVZ retained the capacity for proliferation and migration in response to MPTP-induced Parkinsonism, although the response is less robust than in younger animals. Furthermore, in transgenic mice that overexpress mutant α-synuclein (A53T), brains examined day 21 after MPTP administration showed markedly decreased olfactory bulb and substantia nigra neurogenesis. Our data suggest that in addition to aging effects associated with decline in the number of newly generated cells, mutant α-synuclein reduces MPTP-induced neurogenesis. This could provide a novel therapeutic target for chronic brain repair in this condition.     

Human A53T α-Synuclein Causes Reversible Deficits in Mitochondrial Function and Dynamics in Primary Mouse Cortical Neurons

Parkinson’s disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is α-synuclein (α-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the α-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant α-syn gene develop severe movement disorders. However, the molecular mechanisms of α-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson’s disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T α-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD.     

Receptor-Associated Protein (RAP) Plays a Central Role in Modulating Aβ Deposition in APP/PS1 Transgenic Mice

Background

Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of β-amyloid (Aβ) peptides.

Methodology/Principal Findings

Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05)]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/−)/RAP(+/−) mice correlated with 30–40% increases in amyloid deposition by 9 months of age.

Conclusions/Significance

Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Aβ catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.     

Contrasting Effects of α-Synuclein and γ-Synuclein on the Phenotype of Cysteine String Protein α (CSPα) Null Mutant Mice Suggest Distinct Function of these Proteins in Neuronal Synapses

In neuronal synapses, neurotransmitter-loaded vesicles fuse with presynaptic plasma membrane in a complex sequence of tightly regulated events. The assembly of specialized SNARE complexes plays a pivotal role in this process. The function of the chaperone cysteine string protein α (CSPα) is important for synaptic SNARE complex formation, and mice lacking this protein develop severe synaptic dysfunction and neurodegeneration that lead to their death within 3 months after birth. Another presynaptic protein, α-synuclein, also potentiates SNARE complex formation, and its overexpression rescues the phenotype of CSPα null mutant mice, although these two proteins use different mechanisms to achieve this effect. α-Synuclein is a member of a family of three related proteins whose structural similarity suggests functional redundancy. Here, we assessed whether γ-synuclein shares the ability of α-synuclein to bind synaptic vesicles and ameliorate neurodegeneration caused by CSPα deficiency in vivo. Although the N-terminal lipid-binding domains of the two synucleins showed similar affinity for purified synaptic vesicles, the C-terminal domain of γ-synuclein was not able to interact with synaptobrevin-2/VAMP2. Consequently, overexpression of γ-synuclein did not have any noticeable effect on the phenotype of CSPα null mutant mice. Our data suggest that the functions of α- and γ-synucleins in presynaptic terminals are not fully redundant.     

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.     

TNF-α Producing Innate Lymphoid Cells (ILCs) Are Increased in Active Celiac Disease and Contribute to Promote Intestinal Atrophy in Mice

Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of this study was to characterize the tissue distribution of ILCs in celiac disease (CD), a gluten-driven enteropathy, and analyze their role in gut tissue damage. ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs) isolated from duodenal biopsies of CD patients and healthy controls (CTR) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with peptidoglycan, poly I:C, or CpG, the agonists of TLR2, TLR3, or TLR9 respectively, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Although the percentage of total ILCs did not differ between CD patients and CTR, ILCs producing TNF-α and IFN-γ were more abundant in CD mucosa compared to controls. ILCs expressed TLR2, TLR3 and TLR9 but neither TLR7 nor TLR4. Stimulation of LPMC with poly I:C but not PGN or CpG increased TNF-α and IFN-γ in ILCs. RAG1-deficient mice given poly I:C exhibited increased frequency of TNF-α but not IFN-γ/IL17A-producing ILCs in the gut and depletion of ILCs prevented the poly I:C-driven intestinal damage. Our data indicate that CD-related inflammation is marked by accumulation of ILCs producing TNF-α and IFN-γ in the mucosa. Moreover, ILCs express TLR3 and are functionally able to respond to poly I:C with increased synthesis of TNF-α thus contributing to small intestinal atrophy.     

Investigation of Iron Metabolism in Mice Expressing a Mutant Menke’s Copper Transporting ATPase (Atp7a) Protein with Diminished Activity (Brindled; MoBr /y)

During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [Mo^Br/y]). Mutant mice were rescued by perinatal copper injections, and, after a 7–8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult Mo^Br/y mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived Mo^Br/y mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and Mo^Br/y) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in Mo^Br/y mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, Mo^Br/y mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.     

Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Regulates the Stability and Function of GluA1 α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) Receptor in Neurons

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor abundantly expressed in neurons. Increasing evidence demonstrates that LRP1 regulates synaptic integrity and function at the post synapses, at least partially by regulating glutamate receptors. The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are critical ionotropic glutamate receptors consisting of homotetramer or heterotetramer of GluA1-4 subunits and play an essential role in synaptic transmission and synaptic plasticity. Our previous work has shown that neuronal deletion of the Lrp1 gene in mice leads to decreased level of GluA1 and reduced long-term potentiation. To understand the underlying mechanism, we investigated the cellular and functional consequences of LRP1 deletion in primary neurons. Here, we show that LRP1 interacts with and regulates the cellular distribution and turnover of GluA1. LRP1 knockdown in mouse primary neurons led to accelerated turnover and decreased cell surface distribution of GluA1, which correspond to decreased phosphorylation of GluA1 at S845 and S831 sites. Decreased LRP1 expression also attenuated AMPA-evoked calcium influx and reduced GluA1-regulated neurite outgrowth and filopodia density. Our results reveal a novel mechanism by which LRP1 controls synaptic integrity and function, specifically by regulating GluA1 trafficking, phosphorylation and turnover. They further demonstrate that LRP1-GluA1 pathway may hold promises as a therapeutic target for restoring synaptic functions in neurodegenerative diseases.     

Bacteriophage Lysozyme Synthesis in Escherichia coli K-12(λ) Infected with rII Mutant of Bacteriophage T4

When Escherichia coli K-12(λ) was infected with a T4rII bacteriophage, synthesis of lysozyme appeared at the normal time but stopped 15 min after infection. The lysozyme produced was 1% of the normal level.