Hypocretin (Hcrt) is a neuropeptide synthesized in the lateral hypothalamus (LHT) that plays a key role in maintaining arousal state. In Parkinson's disease (PD), a narcolepsy-like syndrome is commonly seen, and a previous study showed substantial Hcrt neuronal loss in accordance with PD severity. In the present study, we quantitatively examined Hcrt immunoreactivity and α-synuclein and tau pathologies in the LHT and locus coeruleus (LC) in dementia with Lewy bodies (DLB) (n=15), Alzheimer's disease (AD) (n=14), and controls (n=7). In the LHT, substantial Hcrt-positive neurons were detected in controls. In contrast, in DLB and AD, the numbers of both total neurons and Hcrt-positive neurons were significantly reduced. The reduction of the latter was significantly severer in DLB than in AD. In the LC of controls, many Hcrt-positive axonal terminals were found. In contrast, the amount of Hcrt immunoreactivity was significantly reduced both in DLB and AD. In DLB, some Lewy body (LB)-bearing neurons were detected in the LHT, but the Hcrt-positive neurons did not have any LBs. Meanwhile, some tau-positive neurofibrillary tangle (NFT)-bearing neurons were detected in the LHT, and Hcrt-positive neurons occasionally contained NFTs. We observed a significant negative correlation between the number of Hcrt-positive neurons in the LHT and the neurofibrillary stage (r=-0.67, p=0.0067), whereas no significant correlation was found between the number of Hcrt-positive neurons and the Lewy stage (r=-0.47, p=0.077). This is the first report clarifying the substantial loss of Hcrt neurons in the LHT and of Hcrt axonal terminals in the LC in DLB and the correlation between the severity of Hcrt neuronal loss and progression of neurofibrillary pathology.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Lewy bodies and pale bodies in dopaminergic neurons in the substantia nigra are pathological hallmarks of PD. A number of neurodegenerative diseases demonstrate aggregate formation, but how these aggregates are associated with their pathogenesis remains unknown. It has been reported that repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is induced in the nuclei of aged neurons, preserves neuronal function, and protects against neurodegeneration during aging through the repression of cell death-inducing genes. The loss of REST is associated with Alzheimer's disease pathology. However, its function in dopaminergic neurons remains unknown. Here we demonstrated that REST enters the nucleus of aged dopaminergic neurons. On the other hand, REST is partially sequestrated in Lewy bodies and is mostly absent from the nucleus of neurons in brains with PD and dementia with Lewy bodies (DLB). Dopaminergic neuron-specific autophagy-deficient mice exhibit REST accumulation in aggregates. Defects in the protein quality control system induce REST mRNA expression; its gene product mainly appears in aggregates. Our results suggest that Lewy pathology disturbs normal aging processes in dopaminergic neurons by sequestering REST and the loss of REST may associate with the PD pathology.

Immunotherapeutic approaches reducing α-synuclein deposits may provide therapeutic benefit for Dementia with Lewy Bodies (DLB). Immunization with full-length human α-synuclein (hα-Syn) protein in a Parkinson's disease mouse model decreased the accumulation of the aggregated forms of this protein in neurons and reduced neurodegeneration. To enhance the immunogenicity of candidate vaccines and to avoid the risk of autoreactive anti-hα-Syn T-helper (Th) cell responses, we generated three peptide-based epitope vaccines composed of different B-cell epitopes of hα-Syn fused with a "non-self" Th epitope from tetanus toxin (P30). Immunization of mice with these epitope vaccines produced high titers of anti-hα-Syn antibodies that bound to Lewy bodies (LBs) and Lewy neurites (LNs) in brain tissue from DLB cases and induced robust Th cell responses to P30, but not to hα-Syn. Further development of these first generation epitope vaccines may facilitate induction of anti-hα-Syn immunotherapy without producing potentially harmful autoreactive Th cell responses.

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET.

Lewy body dementias are characterized by deposition of alpha-synuclein (α-syn) protein aggregates known as Lewy bodies and Lewy neurites in cortical regions, in addition to brainstem. These aggregates are thought to cause the death of dopaminergic neurons in the substantia nigra and other vulnerable cell types in patients, leading to parkinsonism. There is evidence from mice that localized overexpression of wild-type α-syn leads to dopaminergic cell death in the substantia nigra. However, it is not known how cortical neurons are affected by α-syn. In this study, we used viral overexpression of α-syn to investigate whether localized overexpression within the cortex affects the density, length, and morphology of dendritic spines, which serve as a measure of synaptic connectivity. An AAV2/6 viral vector coding for wild-type human α-syn was used to target overexpression bilaterally to the medial prefrontal cortex within adult mice. After ten weeks the brain was stained using the Golgi-Cox method. Density of dendritic spines in the injected region was increased in layer V pyramidal neurons compared with animals injected with control virus. Immunohistochemistry in separate animals showed human α-syn expression throughout the region of interest, especially in presynaptic terminals. However, phosphorylated α-syn was seen in a discrete number of cells at the region of highest overexpression, localized mainly to the soma and nucleus. These findings demonstrate that at early timepoints, α-syn overexpression may alter connectivity in the cortex, which may be relevant to early stages of the disease. In addition, these findings contribute to the understanding of α-syn, which when overexpressed in the wildtype, non-aggregated state may promote spine formation. Loss of spines secondary to α-syn in cortex may require higher expression, longer incubation, cellular damage, concomitant dopaminergic dysfunction or other two-hit factors to lead to synaptic degeneration.

Parkinson's disease (PD) features selective loss of dopaminergic neurons of the substantia nigra pars compacta accompanied by the accumulation and aggregation of alpha-synuclein in Lewy bodies. PTEN induced putative kinase 1 gene (PINK1) mutations are the second most common genetic cause of autosomal recessive early-onset Parkinson's disease (EOPD). A single nucleotide deletion in PINK1 exon 8 (c.1557delG) was identified in a consanguineous Chinese family with EOPD. The homozygous deletion was co-segregated with disease in the family and resulted in a frameshift after codon 520 with a premature termination at codon 522 (p.K520RfsX3). These findings have significant implications on genetic counseling for the family and may be helpful in considering potential pathogenesis-targeted and disease-modifying strategies which should further improve patient quality of life.

Although the role of epigenetics in Parkinson's disease (PD) has not been extensively studied, α-synuclein, the main component of Lewy bodies, decreased histone H3 acetylation. Here, we determined if there were histone acetylation changes in the primary motor cortex which, according to the Braak model, is one of the last brain regions affected in PD. Net histone H3 acetylation, histone H3 lysine 9 (H3K9), histone H3 lysine 14 (H3K14), histone H3 lysine 18 (H3K18), and histone H3 lysine 23 (H3K23) acetylation was assessed in the primary motor cortex of those affected and unaffected by PD. There was net increase in histone H3 acetylation due to increased H3K14 and H3K18 acetylation. There was a decrease in H3K9 acetylation. No between-groups difference was detected in H3K23 acetylation. Relationships between Unified Lewy Body Staging scores and histone H3 acetylation and substantia nigra depigmentation scores and histone H3 acetylation were observed. No relationships were detected between postmortem interval and histone H3 acetylation and expired age and histone H3 acetylation. These correlational data support the notion that the histone H3 acetylation changes observed here are not due to the postmortem interval or aging. Instead, they are due to PD and/or factors that covary with PD. The data suggest enhanced gene transcription in the primary motor cortex of the PD brain due to increase H3K14 and H3K18 acetylation. This effect is partially offset by a decreased H3K9 acetylation, which might repress gene transcription.

The α7 neuronal nicotinic acetylcholine receptor (α7 nAChR) is a potential target for the development of Parkinson's disease (PD) therapeutics. α-Synuclein (α-Syn), a principal component of Lewy bodies (cytoplasmic inclusions), is a major contributor to PD pathophysiology. Previous studies have demonstrated that activating α7 nAChR protects against nigrostriatal dopamine degeneration in acute and chronic PD animal models induced by 6-hydroxydopamine and rotenone, respectively. In the present study, we investigated the effects of PNU282987, a selective α7 nAChR agonist, against α-Syn-induced neurotoxicity in α-SynWT-, α-SynA30P-, and α-SynE46K-N2a cells. PNU282987 exhibited substantial neuroprotection against both wild-type and mutant-type α-Syn-induced toxicity. Furthermore, PNU282987 promoted transcription factor EB activity and reduced intracellular α-Syn protein levels through autophagy induction. These results highlight the therapeutic potential of α7 nAChR activation in diseases characterized by α-Syn aggregation, such as PD.

Parkinson's disease (PD) is pathologically characterized by intraneuronal α-synuclein (α-Syn) aggregates called Lewy bodies (LBs) as well as the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). On the basis of autopsy studies, Braak et al. hypothesized that Lewy pathology initially occurs in the enteric nervous system, subsequently spreading to the dorsal motor nucleus of the vagus nerve (dmX) and then ascending in the brainstem to the SNpc. However, this hypothetical progression lacks adequate experimental evidence. We previously reported that inoculation of α-Syn preformed fibrils (PFFs) into the gastric wall of wild-type (WT) mice induced LB-like α-Syn aggregates in the dmX via the vagus nerve. However, α-Syn pathology did not spread beyond the dmX up to 12 months postinoculation. In the present study, we inoculated α-Syn PFFs into the gastric wall of bacterial artificial chromosome (BAC) transgenic mice harboring the human α-Syn gene with an A53 T mutation and analyzed the pathology. The transgenic mice had ∼1.5-fold overexpression of α-Syn in the brains and ∼6-fold overexpression of α-Syn in the stomach compared with WT mice. After inoculation of α-Syn PFFs, the transgenic mice developed a higher number of phosphorylated α-Syn (p-α-Syn)-positive neurons in the dmX compared with similarly inoculated WT mice. However, the number of p-α-Syn-positive neurons in the dmX decreased over time, and α-Syn pathology was not observed in other brain regions except in the ambiguous nucleus up to 8 months postinoculation. Taken together, BAC transgenic expression of α-Syn facilitated induction of α-Syn pathology in the brainstem, but not subsequent caudo-rostral spread in accordance with Braak's hypothesis.

The depletion of dopamine in the striatum region and Lewy bodies are the hallmark characteristics of Parkinson's disease. The pathology also includes the upregulation of various Parkinson's disease (PARK) genes and kinases. Two such kinases, LRRK2 and GSK-3β have been directly implicated in the formation of tau and alpha-synuclein proteins, causing PD. Hesperidin (HES) is a flavanone glycoside that has multiple therapeutic benefits including neuroprotective effects. In this study, we examined the neuroprotective effects of HES against 6-hydroxydopamine (6-OHDA) induced-neurotoxicity in the in-vitro and in-vivo model. Hesperidin significantly protected the SH-SY5Y cells' stress against 6-OHDA induced toxicity by downregulating biomarkers of oxidative stress. Furthermore, HES downregulated the kinases lrrk2 and gsk3β along with casp3, casp9, and polg in the zebrafish model. The treatment with HES also improved the locomotor pattern of zebrafish that was affected by 6-OHDA. This study suggests that hesperidin could be a drug of choice in targeting kinases against a 6-OHDA model of PD.

Visual symptoms are relatively common in Parkinson's disease (PD) and optical coherence tomography has indicated possible retinal thinning. Accumulation of aggregated α-synuclein is thought to be a central pathogenic event in the PD brain but there have not as yet been reports of retinal synucleinopathy. Retinal wholemounts were prepared from subjects with a primary clinicopathological diagnosis of PD (N=9), dementia with Lewy bodies (DLB; N=3), Alzheimer's disease (N=3), progressive supranuclear palsy (N=2) as well as elderly normal control subjects (N=4). These were immunohistochemically stained with an antibody against α-synuclein phosphorylated at serine 129, which is a specific molecular marker of synucleinopathy. Phosphorylated α-synuclein-immunoreactive (p-syn IR) nerve fibers were present in 7/9 PD subjects and in 1/3 DLB subjects; these were sparsely distributed and superficially located near or at the inner retinal surface. The fibers were either long and straight or branching, often with multiple en-passant varicosities along their length. The straight fibers most often had an orientation that was radial with respect to the optic disk. Together, these features are suggestive of either retinopetal/centrifugal fibers or of ganglion cell axons. In one PD subject there were sparse p-syn IR neuronal cell bodies with dendritic morphology suggestive of G19 retinal ganglion cells or intrinsically photosensitive ganglion cells. There were no stained nerve fibers or other specific staining in any of the non-PD or non-DLB subjects. It is possible that at least some of the observed visual function impairments in PD subjects might be due to α-synucleinopathy.

Tctex1 is an important structure of dynein light chain in mammalian cells, clarifying the role of Tctex1 in α-synuclein autophagy lysosomal degradation may offer insights into the formation of Lewy bodies and neuronal death. We constructed dsRED-N1-Tctex1 overexpression, pDsRED2-N1-Tctex1(T94E) mutation and transfected into SH-SY5Y cells. Relative protein expression was measured by Western Blot and mRNA levels were measured by real-time quantitative PCR. Confocal microscopy was used to observe their sublocalizations in cells. We found that: WST assay results show that cell activity decreased after Tctex1 mutation (T94E), while Tctex1 overexpression increased cell activity. In Tctex1 mutation transfected cell lines Tctex1 and dynein protein levels decreased; α-synuclein, LC3-II and LAMP2 protein increased. However, α-synuclein, LC3-II and LAMP2 proteins were reduced in Tctex1 overexpression cell lines, with the same trend was found in mRNA levels. In Tctex1 mutation transfected cell lines Tctex1 fluorescence intensity weakened; α-synuclein, LC3-II and LAMP2 fluorescence was enhanced, while α-synuclein, LC3-II and LAMP2 weakened in Tctex1 overexpressing cells. Our results suggest that Tctex1 mutants interference lead to Tctex1 downregulation and dysfunction. Tctex1 overexpression promoted autophagy lysosome fusion and effectively degraded α-synuclein with increased cell activity. Thus, Tctex1 plays an important role in α-synuclein autophagic degradation.

The majority of α-synuclein (α-syn) within Lewy bodies (LBs) has been reported to be phosphorylated at serine 129 (pS129-α-syn), suggesting a central role for phosphorylation in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies. Various studies have investigated the effect of α-syn phosphorylation but have failed to reach a consensus as to whether this modification accelerates or inhibits α-syn aggregation. Nevertheless, pS129-α-syn is a reliable marker of α-syn aggregates and is widely evaluated in biomarkers and post-mortem studies. While several antibodies specific for pS129-α-syn exist, their reactivity with non-specific antigens appears to be a common challenge. To gain valuable insights into the role of α-syn phosphorylation in disease pathogenesis, antibodies that are highly specific to pS129-α-syn are necessary. In this study, we describe the generation of three mouse monoclonal antibodies (mAbs; 5B9, 6H5 and 9G1) using hybridoma technology. These were thoroughly characterized and validated in combination with our previously generated mAb (PS129), and the commercial ab51253 (Abcam). We demonstrated that our mAbs are highly specific for pS129-α-syn and do not cross react with wild-type α-syn. Results from staining of post-mortem human brain tissue showed that our mAbs detect pS129-α-syn pathology in patients with synucleinopathies. This study highlights three new antibodies as excellent and highly specific research tools to explore the role of pS129-α-syn inclusions in synucleinopathies.