Tau knockout exacerbates degeneration of parvalbumin-positive neurons in substantia nigra pars reticulata in Parkinson's disease-related α-synuclein A53T mice.
ABSTRACT: α-Synuclein (α-syn)-induced neurotoxicity has been generally accepted as a key step in the pathogenesis of Parkinson's disease (PD). Microtubule-associated protein tau, which is considered second only to α-syn, has been repeatedly linked with PD in association studies. However, the underlying interaction between these two PD-related proteins in vivo remains unclear. To investigate how the expression of tau affects α-syn-induced neurodegeneration in vivo, we generated triple transgenic mice that overexpressed α-syn A53T mutation in the midbrain dopaminergic neurons (mDANs) with different expression levels of tau. Here, we found that tau had no significant effect on the A53T α-syn-mediated mDANs degeneration. However, tau knockout could modestly promote the formation of α-syn aggregates, accelerate the severe and progressive degeneration of parvalbumin-positive (PV+) neurons in substantia nigra pars reticulata (SNR), accompanied with anxiety-like behavior in aged PD-related α-syn A53T mice. The mechanisms may be associated with A53T α-syn-mediated specifically successive impairment of N-methyl-d-aspartate receptor subunit 2B (NR2B), postsynaptic density-95 (PSD-95) and microtubule-associated protein 1A (MAP1A) in PV+ neurons. Our study indicates that MAP1A may play a beneficial role in preserving the survival of PV+ neurons, and that inhibition of the impairment of NR2B/PSD-95/MAP1A pathway, may be a novel and preferential option to ameliorate α-syn-induced neurodegeneration.
Project description:Transgenic mice line M83 that express the A53T mutant α-synuclein protein at six times the level of endogenous mice α-synuclein are a model of α-synucleinopathy found in Parkinson's disease (PD). This Hualpha-Syn (A53T) PD model is useful in assessing non-motor deficits at earlier stages of onset of PD. We report findings on metabolic changes using [<sup>18</sup>F]FDG PET/CT in the Hualpha-Syn (A53T) PD mouse model in comparison to non-carrier mice. Whole-body PET/CT imaging of male and female mice were carried out 2 h after [<sup>18</sup>F]FDG ip administration under 3% isoflurane anesthesia. Brain images were analyzed with PET images coregistered to a mouse brain MRI template. Hualpha-Syn (A53T) mice had significantly lower [<sup>18</sup>F]FDG uptake in several brain regions compared to the no-carrier mice. Significant hind limb muscle and lower spinal cord [<sup>18</sup>F]FDG hypometabolism at 9 months of age in A53T PD mice was also indicative of neurodegenerative disease, with a progressive motoric dysfunction leading to death. Significant decrease (up to 30%) in [<sup>18</sup>F]FDG uptake were observed in 9-month old male and female Hualpha-Syn (A53) mice. This is consistent with the cortical hypometabolism in PD patients. Hualpha-Syn (A53) mice may thus be a suitable model for studies related to PD α-synucleinopathy for the discovery of new biomarkers.
Project description:Apolipoprotein E (APOE) ?4 genotype is associated with increased risk of dementia in Parkinson's disease (PD), but the mechanism is not clear, because patients often have a mixture of ?-synuclein (?Syn), amyloid-? (A?), and tau pathologies. APOE ?4 exacerbates brain A? pathology, as well as tau pathology, but it is not clear whether APOE genotype independently regulates ?Syn pathology. In this study, we generated A53T ?Syn transgenic mice (A53T) on Apoe knockout (A53T/EKO) or human APOE knockin backgrounds (A53T/E2, E3, and E4). At 12 months of age, A53T/E4 mice accumulated higher amounts of brainstem detergent-insoluble phosphorylated ?Syn compared to A53T/EKO and A53T/E3; detergent-insoluble ?Syn in A53T/E2 mice was undetectable. By immunohistochemistry, A53T/E4 mice displayed a higher burden of phosphorylated ?Syn and reactive gliosis compared to A53T/E2 mice. A53T/E2 mice exhibited increased survival and improved motor performance compared to other APOE genotypes. In a complementary model of ?Syn spreading, striatal injection of ?Syn preformed fibrils induced greater accumulation of ?Syn pathology in the substantia nigra of A53T/E4 mice compared to A53T/E2 and A53T/EKO mice. In two separate cohorts of human patients with PD, APOE ?4/?4 individuals showed the fastest rate of cognitive decline over time. Our results demonstrate that APOE genotype directly regulates ?Syn pathology independent of its established effects on A? and tau, corroborate the finding that APOE ?4 exacerbates pathology, and suggest that APOE ?2 may protect against ?Syn aggregation and neurodegeneration in synucleinopathies.
Project description:Accumulation of misfolded proteins such as amyloid-β (Aβ), tau, and α-synuclein (α-Syn) in the brain leads to synaptic dysfunction, neuronal damage, and the onset of relevant neurodegenerative disorder/s. Dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are characterized by the aberrant accumulation of α-Syn intracytoplasmic Lewy body inclusions and dystrophic Lewy neurites resulting in neurodegeneration associated with inflammation. Cell to cell propagation of α-Syn aggregates is implicated in the progression of PD/DLB, and high concentrations of anti-α-Syn antibodies could inhibit/reduce the spreading of this pathological molecule in the brain. To ensure sufficient therapeutic concentrations of anti-α-Syn antibodies in the periphery and CNS, we developed four α-Syn DNA vaccines based on the universal MultiTEP platform technology designed especially for the elderly with immunosenescence. Here, we are reporting on the efficacy and immunogenicity of these vaccines targeting three B-cell epitopes of hα-Syn aa85-99 (PV-1947D), aa109-126 (PV-1948D), aa126-140 (PV-1949D) separately or simultaneously (PV-1950D) in a mouse model of synucleinopathies mimicking PD/DLB. All vaccines induced high titers of antibodies specific to hα-Syn that significantly reduced PD/DLB-like pathology in hα-Syn D line mice. The most significant reduction of the total and protein kinase resistant hα-Syn, as well as neurodegeneration, were observed in various brain regions of mice vaccinated with PV-1949D and PV-1950D in a sex-dependent manner. Based on these preclinical data, we selected the PV-1950D vaccine for future IND enabling preclinical studies and clinical development.
Project description:Mutations in alpha-synuclein (alpha-Syn) cause Parkinson's disease (PD) in a small number of pedigrees with familial PD. Moreover, alpha-Syn accumulates as a major component of Lewy bodies and Lewy neurites, intraneuronal inclusions that are neuropathological hallmarks of PD. To better understand the pathogenic relationship between alterations in the biology of alpha-Syn and PD-associated neurodegeneration, we generated multiple lines of transgenic mice expressing high levels of either wild-type or familial PD-linked Ala-30 --> Pro (A30P) or Ala-53 --> Thr (A53T) human alpha-Syns. The mice expressing the A53T human alpha-Syn, but not wild-type or the A30P variants, develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Pathologically, affected mice exhibit neuronal abnormalities (in perikarya and neurites) including pathological accumulations of alpha-Syn and ubiquitin. Consistent with abnormal neuronal accumulation of alpha-Syn, brain regions with pathology exhibit increases in detergent-insoluble alpha-Syn and alpha-Syn aggregates. Our results demonstrate that the A53T mutant alpha-Syn causes significantly greater in vivo neurotoxicity as compared with other alpha-Syn variants. Further, alpha-Syn-dependent neurodegeneration is associated with abnormal accumulation of detergent-insoluble alpha-Syn.
Project description:α-Synuclein (α-syn) is a hallmark amyloidogenic protein component of Lewy bodies in dopaminergic neurons affected by Parkinson's disease (PD). Despite the multi-faceted gene regulation of α-syn in the nucleus, the mechanism underlying α-syn crosstalk in chromatin remodeling in PD pathogenesis remains elusive. Here, we identified transcriptional adapter 2-alpha (TADA2a) as a novel binding partner of α-syn using the BioID system. TADA2a is a component of the p300/CBP-associated factor and is related to histone H3/H4 acetylation. We found that α-syn A53T was more preferentially localized in the nucleus than the α-syn wild-type (WT), leading to a stronger disturbance of TADA2a. Indeed, α-syn A53T significantly reduced the level of histone H3 acetylation in SH-SY5Y cells; its reduction was also evident in the striatum (STR) and substantia nigra (SN) of mice that were stereotaxically injected with α-syn preformed fibrils (PFFs). Interestingly, α-syn PFF injection resulted in a decrease in TADA2a in the STR and SN of α-syn PFF-injected mice. Furthermore, the levels of TADA2a and acetylated histone H3 were significantly decreased in the SN of patients with PD. Therefore, histone modification through α-syn A53T-TADA2a interaction may be associated with α-syn-mediated neurotoxicity in PD pathology.
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: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:A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids-long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCA A53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1-122 and 1-90 from the SNCA A53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.
Project description:Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (?-syn). Duplication, triplication or genetic mutations in ?-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., ?-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of ?-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T ?-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1-2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments similar to PD patients and may provide useful endpoints for assessing novel therapeutic interventions for PD.
Project description:The motor and nonmotor symptoms of PD involve several brain regions. However, whether ?-syn pathology originating from the SNc can directly lead to the pathological changes in distant cerebral regions and induce PD-related symptoms remains unclear. Here, AAV9-synapsin-mCherry-human SNCA (A53T) was injected into the unilateral SNc of mice. Motor function and olfactory sensitivity were evaluated. Our results showed that AAV9-synapsin-mCherry-human SNCA was continuously expressed in SNc. The animals showed mild motor and olfactory dysfunction at 7 months after viral injection. The pathology in SNc was characterized by the loss of dopaminergic neurons accompanied by ER stress. In the striatum, h?-syn expression was high, CaMK?-2 and NR2B expression decreased, and active synapses reduced. In the olfactory bulb, h?-syn expression was high, and aging cells in the mitral layer increased. The results suggested that h?-syn was transported in the striatum and OB along the nerve fibers that originated from the SNc and induced pathological changes in the distant cerebral regions, which contributed to the motor and nonmotor symptoms of PD.