The substitution of Proline with Serine at residue 56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) has been linked to an atypical autosomal dominant form of familial amyotrophic lateral sclerosis 8 (ALS8). To investigate the pathogenic mechanism of P56S VAPB in ALS, we generated transgenic (Tg) mice that heterologously express human wild-type (WT) and P56S VAPB under the control of a pan-neuronal promoter Thy1.2. While WT VAPB Tg mice did not exhibit any overt motor behavioral phenotypes, P56S VAPB Tg mice developed progressive hyperactivities and other motor abnormalities. VAPB protein was accumulated as large punctate in the soma and proximal dendrites of both corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs) in P56S VAPB Tg mice. Concomitantly, a significant increase of endoplasmic reticulum stress and unfolded protein response and the resulting up-regulation of pro-apoptotic factor CCAAT/enhancer-binding protein homologous protein expression were observed in the CSMNs and SMNs of P56S VAPB Tg mice. However, only a progressive loss of CSMNs but not SMNs was found in P56S VAPB Tg mice. In SMNs, P56S VAPB promoted a rather selective translocation of VAPB protein onto the postsynaptic site of C-boutons that altered the morphology of C-boutons and impaired the spontaneous rhythmic discharges of SMNs. Therefore, these findings provide new pathophysiological mechanisms of P56S VAPB that differentially affect the function and survival of CSMNs and SMNs in ALS8.
α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.
In the hippocampus, activation of nicotinic receptors that include α4 and β2 subunits (α4β2*) facilitates memory formation. α4β2* receptors may also play a role in nicotine withdrawal, and their loss may contribute to cognitive decline in aging and Alzheimer’s disease (AD). However, little is known about their cellular function in the hippocampus. Therefore, using optogenetics, whole cell patch clamping and voltage-sensitive dye (VSD) imaging, we measured nicotinic excitatory postsynaptic potentials (EPSPs) in hippocampal CA1. In a subpopulation of inhibitory interneurons, release of ACh resulted in slow depolarizations(rise time constant 33.2 ± 6.5 ms, decay time constant 138.6 ± 27.2 ms)mediated by the activation of α4β2* nicotinic receptors. These interneurons had somata and dendrites located in the stratum oriens(SO) and stratum lacunosum-moleculare (SLM). Furthermore, α4β2* nicotinic EPSPs were largest in the SLM. Thus, our data suggest that nicotinic EPSPs in hippocampal CA1 interneuronsare predominantly mediated by α4β2* nicotinic receptors and their activation may preferentially affect extrahippocampal inputs in SLM of hippocampal CA1.
Inhibitory interneuron; nicotinic excitatory postsynaptic potential; hippocampus; voltage-sensitive dye imaging; optogenetics
Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-β1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-β1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-β1 had balancing effects on mast cell survival, migration, and FcεRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-β1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-β1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.
Mutations in α-synuclein and Leucine-rich repeat kinase 2 (LRRK2) are linked to autosomal dominant forms of Parkinson’s disease (PD). However, little is known about any potential pathophysiological interplay between these two PD-related genes. Here we show in transgenic mice that although over-expression of LRRK2 alone did not cause neurodegeneration, the presence of excess LRRK2 greatly accelerated the progression of neuropathological abnormalities developed in PD-related A53T α-synuclein transgenic mice. Moreover, we found that LRRK2 promoted the abnormal aggregation and somatic accumulation of α-synuclein in A53T mice, likely resulted from the impairment of microtubule dynamics, Golgi organization, and ubiquitin-proteasome pathway. Conversely, genetic ablation of LRRK2 preserved the Golgi structure, suppressed the aggregation and somatic accumulation of α-synuclein, and thereby delayed the progression of neuropathology in A53T mice. These findings demonstrate that over-expression of LRRK2 enhances α-synuclein-mediated cytotoxicity and suggest inhibition of LRRK2 expression as a potential therapeutic option for ameliorating α-synuclein-induced neurodegeneration.
LRRK2; G2019S; α-synuclein; A53T; Golgi apparatus; microtubule; ubiquitin; mitochondria; aggregation; transgenic; knockout; Parkinson’s disease
This paper demonstrates the role of R7 RGS proteins in the timing of the ERG b-wave. The data suggest that R7 RGS proteins are important components in the metabotropic glutamate receptor 6 (mGluR6) signaling pathway in ON-bipolar cells in the retina.
In the Gβ5−/− mouse, the electroretinogram (ERG) b-wave is absent, and the R7 subfamily of regulators of G protein signaling (RGS), which includes RGS6, -7, -9, and -11, is downregulated. Mutant mouse strains deficient in RGS7 or -11 were characterized, and the SG711 strain which is deficient in both proteins was examined, to learn whether the loss of some of these RGS proteins causes the absence of the ERG b-wave.
Antibodies to RGS7 and -11 were generated to determine their expression levels and localizations in retinas with various genetic backgrounds by Western blot analysis and immunohistochemistry, respectively. The implicit times and amplitudes of ERG a- and b-waves were analyzed to examine photoreceptor and bipolar cell functions.
RGS7 and -11 co-localized to the dendritic tips of the ON-bipolar cells. In the RGS11−/− mouse, the level of RGS7 protein increased. However, the level of RGS11 protein remained unchanged in the RGS7 mutant mouse, where a truncated RGS7 protein was expressed due to the deletion of exon 10. In the SG711 mouse retina, the Gβ5-S protein level was reduced. The ERG b-wave of SG711 mice was markedly delayed. In contrast, RGS11−/− mice showed a moderately delayed b-wave, whereas the RGS7 mutant mice showed normal ERG responses.
The data demonstrate the presence of a delayed ERG b-wave in SG711 mice and a functionally redundant role for RGS11 and -7 at the tips of ON-bipolar cell dendrites. These results suggest that RGS11 or -7 works as the major physiological GAP (GTPase acceleration protein) for Gαo1 in ON-bipolar cells.
Dysfunction of alsin, particularly its putative Rab5 guanine-nucleotide-exchange factor activity, has been linked to one form of juvenile onset recessive familial amyotrophic lateral sclerosis (ALS2). Multiple lines of alsin knockout (ALS2-/-) mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in ALS2-/- neurons. To directly examine the Rab5-mediated endosomal trafficking in ALS2-/- neurons, we introduced green fluorescent protein (GFP)-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes. Here we report that Rab5-mediated endocytosis was severely altered in ALS2-/-neurons. Excessive accumulation of Rab5-positive vesicles was observed in ALS2-/- neurons, which correlated with a significant reduction in endosomal motility and augmentation in endosomal conversion to lysosomes. Consequently, a significant increase in endosome/lysosome-dependent degradation of internalized glutamate receptors was observed in ALS2-/- neurons. These phenotypes closely resembled the endosomal trafficking abnormalities induced by a constitutively active form of Rab5 in wild-type neurons. Therefore, our findings reveal a negatively regulatory mechanism of alsin in Rab5-mediated endosomal trafficking, suggesting that enhanced endosomal degradation in ALS2-/- neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases.
β-site APP cleavage enzyme 1 (BACE1) is the β-secretase responsible for generating amyloid-β (Aβ) peptides in Alzheimer’s disease (AD). Previous studies suggest that activation of protein kinase C (PKC) modulates the β-secretase-mediated cleavage of APP and reduces the production of Aβ. The mechanism of PKC-mediated modulation of β-secretase activity, however, remains elusive. We report here that activation of PKC modulated β-secretase activity through either suppressing the accumulation or promoting the translocation of BACE1 protein in a cell type-dependent manner. We found that activation of PKC suppressed the accumulation of BACE1 protein in fibroblasts through an enhancement of intracellular protease activities. In neurons, activation of PKC did not alter the expression level of BACE1, but led to more BACE1 translocated to the cell surface, resulting in a decreased cleavage of APP at the β1 site. Together, Our findings provide novel mechanisms of PKC-mediated modulation of β-secretase activity, suggesting that alteration of the intracellular trafficking of BACE1 may serve as a useful therapeutic strategy to lower the production of Aβ in AD.
BACE1; APP; PKC; protein degradation; protein translocation; amyloid β; β-secretase; fibroblast; neuron
Parkinson’s disease (PD), a progressive neurodegenerative disease characterized by bradykinesia, rigidity, and resting tremor, is the most common neurodegenerative movement disorder. Although the majority of PD cases are sporadic, some are inherited, including those caused by leucine-rich repeat kinase 2 (LRRK2) mutations. The substitution of serine for glycine at position 2019 (G2019S) in the kinase domain of LRRK2 represents the most prevalent genetic mutation in both familial and apparently sporadic cases of PD. Because mutations in LRRK2 are likely associated with a toxic gain of function, destabilization of LRRK2 may be a novel way to limit its detrimental effects. Here we show that LRRK2 forms a complex with heat shock protein 90 (Hsp90) in vivo and that inhibition of Hsp90 disrupts the association of Hsp90 with LRRK2 and leads to proteasomal degradation of LRRK2. Hsp90 inhibitors may therefore limit the mutant LRRK2-elicited toxicity to neurons. As a proof of principle, we show that Hsp90 inhibitors rescue the axon growth retardation caused by overexpression of the LRRK2 G2019S mutation in neurons. Therefore, inhibition of LRRK2 kinase activity can be achieved by blocking Hsp90-mediated chaperone activity and Hsp90 inhibitors may serve as potential anti-PD drugs.
Hsp90; LRRK2; G2019S; Parkinson’s disease; protein degradation; chaperone
Autosomal recessive mutations in the ALS2 gene have been linked to juvenile-onset amyotrophic lateral sclerosis (ALS2), primary lateral sclerosis and juvenile-onset ascending hereditary spastic paraplegia. Except for two recently identified missense mutations, all other mutations in the ALS2 gene lead to a premature stop codon and likely abrogate all the potential functions of alsin, the protein encoded by the ALS2 gene. To study the pathologic mechanisms of ALS2 deficiency, four different lines of ALS2 knockout (ALS2−/−) mice have been generated by independent groups. The loss of ALS2/alsin does not have a drastic effect on the survival or function of motor neurons in mice. However, subtle deficits observed in the behavior and pathology of these mice have aided in our understanding of the relationship between alsin and motor neuron dysfunction. In this review, we summarize and reconcile major findings of ALS2−/− mice and attempt to place these results within the larger context of modeling recessive movement disorders in mice.
Amyotrophic lateral sclerosis; ALS2; Alsin; Knockout mice; Mouse model; Guanine nucleotide exchange factor; Primary lateral sclerosis; Hereditary spastic paraplegia
Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2-/- spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2-/- neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2-/- neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.
ALS2; knock-out mouse; motor neuron; GRIP1; AMPA receptor; excitotoxicity
Autosomal recessive mutations in the ALS2 gene have been linked to juvenile-onset amyotrophic lateral sclerosis (ALS2), primary lateral sclerosis and juvenile-onset ascending hereditary spastic paraplegia. Except for two recently identified missense mutations, all other mutations in the ALS2 gene lead to a premature stop codon and likely abrogate all the potential functions of alsin, the protein encoded by the ALS2 gene. To study the pathologic mechanisms of ALS2 deficiency, four different lines of ALS2 knockout (ALS2–/–) mice have been generated by independent groups. The loss of ALS2/alsin does not have a drastic effect on the survival or function of motor neurons in mice. However, subtle deficits observed in the behavior and pathology of these mice have aided in our understanding of the relationship between alsin and motor neuron dysfunction. In this review, we summarize and reconcile major findings of ALS2–/– mice and attempt to place these results within the larger context of modeling recessive movement disorders in mice.
Amyotrophic lateral sclerosis; ALS2; Alsin; Knockout mice; Mouse model; Guanine nucleotide exchange factor; Primary lateral sclerosis; Hereditary spastic paraplegia
Dysfunction of the ALS2 gene has been linked to one form of juvenile onset autosomal recessive amyotrophic lateral sclerosis (ALS). Previous in vitro studies suggest that over-expression of ALS2 protects cells from mutant Cu/Zn superoxide dismutase (SOD1)-induced cytotoxicity. To test whether ALS2 plays a protective role against mutant SOD1-mediated motor neuron degeneration in vivo, we examined the progression of motor neuron disease in SOD1G93A mice on an ALS2 null background. Our data suggest that deficiency in the ALS2 gene does not affect the pathogenesis of SOD1G93A mice.
Amyotrophic lateral sclerosis (ALS); ALS2; Alsin; SOD1; SOD1G93A mice
The G59S missense mutation at the conserved microtubule-binding domain of p150glued, a major component of dynein/dynactin complex, has been linked to an autosomal dominant form of motor neuron disease (MND). To study how this mutation affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration, we generated p150glued G59S knock-in mice. We found that the G59S mutation destabilizes p150glued and disrupts the function of dynein/dynactin complex, resulting in early embryonic lethality of homozygous knock-in mice. Heterozygous knock-in mice, which developed normally, displayed MND-like phenotypes after 10 months of age, including excessive accumulation of cytoskeletal and synaptic vesicle proteins at neuromuscular junctions, loss of spinal motor neurons, increase of reactive astrogliosis, and shortening of gait compared with wild-type littermates and age-matched p150glued heterozygous knock-out mice. Our findings indicate that the G59S mutation in p150glued abrogates the normal function of p150glued and accelerates motor neuron degeneration.
dynactin; dynein; p150glued; motor neuron disease; mouse model; ALS
Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is caused by a selective loss of motor neurons in the CNS. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knock-out (ALS2−/−) mice. Although ALS2−/− mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2−/− mice showed a higher anxiety response in the open-field and elevated plus-maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease by 20 months of age, ALS2−/− mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared with wild-type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model but predisposes neurons to oxidative stress.
ALS2; knock-out mouse; motor neuron; motor coordination; motor learning; oxidative stress
One form of juvenile onset autosomal recessive amyotrophic lateral sclerosis (ALS2) has been linked to the dysfunction of the ALS2 gene. The ALS2 gene is expressed in lymphoblasts, however, whether ALS2-deficiency affects periphery blood is unclear. Here we report that ALS2 knockout (ALS2−/−) mice developed peripheral lymphopenia but had higher proportions of hematopoietic stem and progenitor cells in which the stem cell factor-induced cell proliferation was up-regulated. Our findings reveal a novel function of the ALS2 gene in the lymphopoiesis and hematopoiesis, suggesting that the immune system is involved in the pathogenesis of ALS2.
amyotrophic lateral sclerosis (ALS); ALS2; ALS2 knockout mice; lymphopenia; hematopoietic stem and progenitor cells; cytokine-stimulated proliferation; stroma