BPOZ-2 Gene Delivery Ameliorates Alpha-Synucleinopathy in A53T Transgenic Mouse Model of Parkinson's Disease.
ABSTRACT: Ankyrin-rich BTB/POZ domain containing protein-2 or BPOZ-2, a scaffold protein, has been recently shown to control the degradation of many biological proteins ranging from embryonic development to tumor progression. However, its role in the process of neuronal diseases has not been properly explored. Since, abnormal clearance of metabolic proteins contributes to the development of alpha-synuclein (?-syn) pathologies in Parkinson's disease (PD), we are interested to explore if BPOZ-2 participates in the amelioration of ?-syn in vivo in basal ganglia. Here we report that lentiviral administration of bpoz-2 gene indeed lowers the burden of ?-syn in DA neurons in the nigra of A53T transgenic (A53T-Tg) mouse. Our detailed immunological analyses have shown that the overexpression of bpoz-2 dramatically improves both somatic and neuritic ?-syn pathologies in the nigral DA neurons. Similarly, the specific ablation of bpoz-2 by lentiviral-shRNA stimulates the load of monomeric and polymeric forms of ?-syn in the nigral DA neurons of A53T-Tg. While investigating the mechanism, we observed that BPOZ-2 was involved in a protein-protein association with PINK1 and therefore could stimulate PINK1-dependent autophagic clearance of ?-syn. Our results have demonstrated that bpoz-2 gene delivery could have prospect in the amelioration of alpha-synucleinopathy in PD and other Lewy body diseases.
Project description:Parkinson's disease (PD) is an age-dependent neurodegenerative disease that can be caused by genetic mutations in ?-synuclein (?-syn) or duplication of wild-type ?-syn; PD is characterized by the deposition of ?-syn aggregates, indicating a gain of toxicity from accumulation of ?-syn. Although the major neuropathologic feature of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra, non-motor symptoms including anxiety, cognitive defect and sleep disorder precede the onset of motor impairment, and many clinical symptoms of PD are not caused by degeneration of DA neurons. Non-human primate models of PD are important for revealing the early pathology in PD and identifying effective treatments. We established transgenic PD rhesus monkeys that express mutant ?-syn (A53T). Six transgenic A53T monkeys were produced via lentiviral vector expressing A53T in fertilized monkey eggs and subsequent embryo transfer to surrogates. Transgenic A53T is expressed in the monkey brain and causes age-dependent non-motor symptoms, including cognitive defects and anxiety phenotype, without detectable sleeping disorders. The transgenic ?-syn monkeys demonstrate the specific early symptoms caused by mutant ?-syn and provide insight into treatment of early PD.
Project description:Increasing the function of residual dopaminergic neurons in the nigra of PD patients is an important area of research as it may eventually compensate the loss. Although tyrosine hydroxylase (TH) is the rate-limiting enzyme in the dopamine (DA) biosynthesis pathway, there are no effective drugs/molecules to upregulate TH and increase the production of DA in nigral dopaminergic neurons. This study underlines the importance of aspirin in stimulating the expression of TH and increasing the level of DA in dopaminergic neurons. At low doses, aspirin increased the expression of TH and the production of DA in mouse MN9D dopaminergic neuronal cells. Accordingly, oral administration of aspirin increased the expression of TH in the nigra and upregulated the level of DA in striatum of normal C57/BL6 mice and aged A53T ?-syn transgenic mice. Oral aspirin also improved locomotor activities of normal mice and A53T transgenic mice. While investigating mechanisms, we found the presence of cAMP response element (CRE) in the promoter of TH gene and the rapid induction of cAMP response element binding (CREB) activation by aspirin in dopaminergic neuronal cells. Aspirin treatment also increased the level of phospho-CREB in the nigra of C57/BL6 mice. The abrogation of aspirin-induced expression of TH by siRNA knockdown of CREB and the recruitment of CREB to the TH gene promoter by aspirin suggest that aspirin stimulates the transcription of TH in dopaminergic neurons via CREB. These results highlight a new property of aspirin in stimulating the TH-DA pathway, which may be beneficial in PD patients. Graphical Abstract ?.
Project description:Transgenic (Tg) mouse models of Parkinson's disease (PD) generated to date have primarily been designed to overexpress human alpha-synuclein (alpha-syn) to recapitulate PD-like motor impairments as well as PD-like nigrostriatal degeneration and alpha-syn pathology. However, cognitive impairments and cortical alpha-syn pathology are also common in PD patients. To model these features of PD, we created forebrain-specific conditional Tg mice that overexpress human wild type (WT) or A53T mutant alpha-syn. Here we show that both WT and A53T mutant alpha-syn lead to massive degeneration of postmitotic neurons in the hippocampal dentate gyrus (DG) during postnatal development, with hippocampal synapse loss as evidenced by reduced levels of pre- and postsynaptic markers. However, when mutant and WT alpha-syn expression was repressed until the Tg mice were mature postnatally and then induced for several months, no hippocampal neuron loss was observed. These data imply that developing neurons are more vulnerable to degenerate than mature neurons as a consequence of forebrain WT and mutant alpha-syn overexpression.
Project description:Neurosin is a predominant serine protease in the central nervous system (CNS) and has been shown to play a role in the clearance of ?-synuclein (?-syn) which is centrally involved in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Although it has been previously shown that neurosin and ?-syn colocalize and that neurosin degrades ?-syn aggregates in vitro, it is not clear if neurosin is dysregulated in the brains of patients with PD/DLB and to what extent delivery of neurosin into the CNS might ameliorate the deficits associated with ?-syn accumulation in vivo. We analyzed the levels of neurosin in the brains of patients with PD/DLB and in ?-syn transgenic (tg) models. With increased accumulation of ?-syn, we observed decreased neurosin expression. Lentiviral vector (LV) driven expression of neurosin in neuronal cell cultures reduced the accumulation of wild type but not A53T ?-syn and prevented ?-syn associated toxicity. Neuropathological analysis following delivery of LV-Neurosin to ?-syn tg mice resulted in reduced accumulation of ?-syn and reversal of neurodegenerative alterations in wild type but not A53T ?-syn tg mice. Therefore, viral vector driven expression of neurosin may warrant further investigation as a potential therapeutic tool for DLB.
Project description:α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson's disease (PD). Point mutations and multiplications of the α-Syn, which encodes the <i>SNCA</i> gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4-5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies.
Project description:Human studies and preclinical models of Parkinson's disease implicate the involvement of both the innate and adaptive immune systems in disease progression. Further, pro-inflammatory markers are highly enriched near neurons containing pathological forms of alpha synuclein (?-syn), and ?-syn overexpression recapitulates neuroinflammatory changes in models of Parkinson's disease. These data suggest that ?-syn may initiate a pathological inflammatory response, however the mechanism by which ?-syn initiates neuroinflammation is poorly understood. Silencing endogenous ?-syn results in a similar pattern of nigral degeneration observed following ?-syn overexpression. Here we aimed to test the hypothesis that loss of ?-syn function within nigrostriatal neurons results in neuronal dysfunction, which subsequently stimulates neuroinflammation. Adeno-associated virus (AAV) expressing an short hairpin RNA (shRNA) targeting endogenous ?-syn was unilaterally injected into the substantia nigra pars compacta (SNc) of adult rats, after which nigrostriatal pathology and indices of neuroinflammation were examined at 7, 10, 14 and 21 days post-surgery. Removing endogenous ?-syn from nigrostriatal neurons resulted in a rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) within transduced nigral neurons. Nigral MHC-1 expression occurred prior to any overt cell death and coincided with the recruitment of reactive microglia and T-cells to affected neurons. Following the induction of neuroinflammation, ?-syn knockdown resulted in a 50% loss of nigrostriatal neurons in the SNc and a corresponding loss of nigrostriatal terminals and dopamine (DA) concentrations within the striatum. Expression of a control shRNA did not elicit any pathological changes. Silencing ?-syn within glutamatergic neurons of the cerebellum did not elicit inflammation or cell death, suggesting that toxicity initiated by ?-syn silencing is specific to DA neurons. These data provide evidence that loss of ?-syn function within nigrostriatal neurons initiates a neuronal-mediated neuroinflammatory cascade, involving both the innate and adaptive immune systems, which ultimately results in the death of affected neurons.
Project description:Accumulation of human wild-type (wt) α-synuclein (α-syn) induces neurodegeneration in humans and in experimental rodent models of Parkinson disease (PD). It also leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). We overexpressed glucose regulated protein 78, also known as BiP (GRP78/BiP), to test the hypothesis that this ER chaperone modulates the UPR, blocks apoptosis, and promotes the survival of nigral dopamine (DA) neurons in a rat model of PD induced by elevated level of human α-syn. We determined that α-syn activates ER stress mediators associated with pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) signaling pathways as well as proaoptotic CCAAT/-enhancer-binding protein homologous protein (CHOP) in nigral DA neurons. At the same time, overexpression of GRP78/BiP diminished α-syn neurotoxicity by down regulating ER stress mediators and the level of apoptosis, promoted survival of nigral tyrosine hydroxylase (TH) positive cells and resulted in higher levels of striatal DA, while eliminating amphetamine induced behavioral asymmetry. We also detected a complex between GRP78/BiP and α-syn that may contribute to prevention of the neurotoxicity caused by α-syn. Our data suggest that the molecular chaperone GRP78/BiP plays a neuroprotective role in α-syn-induced Parkinson-like neurodegeneration.
Project description:Overexpression or mutation of ?-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]?-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]?-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]?-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]?-Synuclein-induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]?-Synuclein-induced sensitivity to oxidative stress treatment. [A53T]?-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]?-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]?-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to ?-Synuclein.
Project description:Both ?-Synuclein (?Syn) accumulation and mitochondrial dysfunction have been implicated in the pathology of Parkinson's disease (PD). Although studies suggest that ?Syn and its missense mutant, A53T, preferentially accumulate in the mitochondria, the mechanisms by which ?Syn and mitochondrial proteins regulate each other to trigger mitochondrial and neuronal toxicity are poorly understood. ATP-dependent Clp protease (ClpP), a mitochondrial matrix protease, plays an important role in regulating mitochondrial protein turnover and bioenergetics activity. Here, we show that the protein level of ClpP is selectively decreased in ?Syn-expressing cell culture and neurons derived from iPS cells of PD patient carrying ?Syn A53T mutant, and in dopaminergic (DA) neurons of ?Syn A53T mice and PD patient postmortem brains. Deficiency in ClpP induces an overload of mitochondrial misfolded/unfolded proteins, suppresses mitochondrial respiratory activity, increases mitochondrial oxidative damage and causes cell death. Overexpression of ClpP reduces ?Syn-induced mitochondrial oxidative stress through enhancing the level of Superoxide Dismutase-2 (SOD2), and suppresses the accumulation of ?Syn S129 phosphorylation and promotes neuronal morphology in neurons derived from PD patient iPS cells carrying ?Syn A53T mutant. Moreover, we find that ?Syn WT and A53T mutant interact with ClpP and suppress its peptidase activity. The binding of ?Syn to ClpP further promotes a distribution of ClpP from soluble to insoluble cellular fraction in vitro and in vivo, leading to reduced solubility of ClpP. Compensating for the loss of ClpP in the substantia nigra of ?Syn A53T mice by viral expression of ClpP suppresses mitochondrial oxidative damage, and reduces ?Syn pathology and behavioral deficits of mice. Our findings provide novel insights into the mechanism underlying ?Syn-induced neuronal pathology, and they suggest that ClpP might be a useful therapeutic target for PD and other synucleinopathies.
Project description:Mutations, duplication and triplication of ?-synuclein genes are linked to familial Parkinson's disease (PD), and aggregation of ?-synuclein (?-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of ?-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type ?-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-?-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the ?-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing ?-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of ?-syn after the combined insults. In summary, these results reveal that the overexpressed ?-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of ?-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.