Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

The olfactory bulb (OB) seems to be the first affected structure in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Lewy body dementia (LBD). Deposits of protein aggregates, increased dopaminergic neurons, and decreased cholinergic inputs have all been described in the OB of these diseases. We investigated here the contribution of the activated microglial cells to the increased deposits of protein aggregates. We quantified the number of activated microglial cells and astrocytes in the OB of patients with histological diagnosis of PD (n= 5), AD (n= 13), and LBD (n= 7) and aged-matched controls (n= 8). Specific consensus diagnostic criteria were applied for AD, LBD, and PD. Protein aggregates were scored in the OB as grade 0, none; grade 1, mild; grade 2, moderate; and grade 3, severe. OB sections from the 33 subjects were stained with specific antibodies markers for reactive astrocytes (GFAP) and microglial cells (Iba1 and HLA-DR). The total number of Iba1-ir (Iba-immunoreactive) and HLAD-DR cells was estimated by stereological analysis, while quantification of astrocytes was performed by GFAP optical density. Statistical analysis was done using the Stata 12.0 software. The number of microglia and activated microglia cells (HLA-RD-ir) was increased in patients with neurodegenerative diseases (p< 0.05). Moreover, the density of GFAP-ir cells was higher in the OB of patients. Neither the number of microglia cells nor the density of astrocytes correlated with the number of b-amyloid and alpha-synuclein deposits, but the density of Iba1-ir cells correlated with the number of p-Tau aggregates. Activated microglial cells and reactive astrocytes are present in the OB of patients with neurodegenerative diseases. The lack of correlation between the number of activated microglia cells and protein deposits indicate that they might independently contribute to the degenerative process. The presence of microglia is related to phosphorylated Tau deposits in neurodegenerative diseases.

Protein misfolding diseases refer to a variety of disorders that develop as a consequence of the misfolding of proteins in various organs. The etiologies of Parkinson's and Alzheimer's disease remain unclear, but it seems that type two diabetes and other prediabetic states could contribute to the appearance of the sporadic forms of these diseases. In addition to amylin deposition, other amyloidogenic proteins implicated in the pathophysiology of neurodegenerative diseases could have important roles in the pathogenesis of this disease. As we have previously demonstrated the presence of alpha-synuclein deposits in the pancreas of patients with synucleinopathies, as well as tau and A beta deposits in the pancreatic tissue of Alzheimer's disease patients, we studied the immunoreactivity of amylin, tau and alpha-synuclein in the pancreas of 138 subjects with neurodegenerative diseases or type two diabetes and assessed whether the pancreatic beta-cells of these subjects present cooccurrence of misfolded proteins. Furthermore, we also assessed the pancreatic expression of prion protein (PrP) in these subjects and its interaction, both in the pancreas and brain, with alpha-synuclein, tau, A beta and amylin. Our study shows, for the first time, that along with amylin, pancreatic alpha-synuclein, A beta, PrP and tau may contribute together to the complex pathophysiology of type two diabetes and in the appearance of insulin resistance in Alzheimer's and Parkinson's disease. Furthermore, we show that the same mixed pathologies that are observed in the brains of patients with neurodegenerative diseases are also present outside the nervous system. Finally, we provide the first histological evidence of an interaction between PrP and A beta, alpha-synuclein, amylin or tau in the pancreas and locus coeruleus. These findings will shed more light on the common pathological pathways shared by neurodegenerative diseases and type two diabetes, benefiting the exploration of common therapeutic strategies to prevent or treat these devastating amyloid diseases.

Parkinson's disease is characterized by motor and nonmotor symptoms that gradually appear as a consequence of the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Currently, no treatment can slow Parkinson's disease progression. Inasmuch, there is a need to develop animal models that can be used to understand the pathophysiological mechanisms underlying dopaminergic neuron death. The initial goal of this study was to determine if canine adenovirus type 2 (CAV-2) vectors are effective gene transfer tools in the monkey brain. A second objective was to explore the possibility of developing a large nonhuman primate that expresses one of the most common genetic mutations causing Parkinson's disease. Our studies demonstrate the neuronal tropism, retrograde transport, biodistribution, and efficacy of CAV-2 vectors expressing GFP and leucine-rich repeat kinase 2 (LRRK2(G2019S)) in the Macaca fascicularis brain. Our data also suggest that following optimization CAV-2-mediated LRRK2(G2019S) expression could help us model the neurodegenerative processes of this genetic subtype of Parkinson's disease in monkeys.

Objective Alzheimer disease (AD) is the leading cause of dementia, and although its etiology remains unclear, it seems that type 2 diabetes mellitus (T2DM) and other prediabetic states of insulin resistance could contribute to the appearance of sporadic AD. As such, we have assessed whether tau and beta-amyloid (A beta) deposits might be present in pancreatic tissue of subjects with AD, and whether amylin, an amyloidogenic protein deposited in the pancreas of T2DM patients, might accumulate in the brain of AD patients. Methods We studied pancreatic and brain tissue from 48 individuals with no neuropathological alterations and from 87 subjects diagnosed with AD. We examined A beta and tau accumulation in the pancreas as well as that of amylin in the brain. Moreover, we performed proximity ligation assays to ascertain whether tau and/or A beta interact with amylin in either the pancreas or brain of these subjects. Results Cytoplasmic tau and A beta protein deposits were detected in pancreatic beta cells of subjects with AD as well as in subjects with a normal neuropathological examination but with a history of T2DM and in a small cohort of control subjects without T2DM. Furthermore, we found amylin deposits in the brain of these subjects, providing histological evidence that amylin can interact with A beta and tau in both the pancreas and hippocampus. Interpretation The presence of both tau and A beta inclusions in pancreatic beta cells, and of amylin deposits in the brain, provides new evidence of a potential overlap in the mechanisms underlying the pathogenesis of T2DM and AD. ANN NEUROL 2019

Purpose Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces degeneration of dopaminergic neurons and reproduces the motor features of Parkinson disease (PD); however, the effect of MPTP on extranigral structures has been poorly studied. The aim of this research was to study the cardiac sympathetic innervation of control and MPTP-treated monkeys in order to describe the influence of MPTP toxicity on cardiac tissue. Methods Eight monkeys were included in the study and divided into two groups, four monkeys serving as controls and four forming the MPTP group. Sections from the anterior left ventricle were immunohistochemically examined to characterize the sympathetic fibers of cardiac tissue. The intensity of immunoreactivity in the nerve fibers was quantitatively analyzed using ImageJ software. Results As occurs in PD, the sympathetic peripheral nervous system is affected in MPTP-treated monkeys. The percentage of tyrosine hydroxylase immunoreactive fibers in the entire fascicle area was markedly lower in the MPTP group (24.23%) than the control group (35.27%) (p < 0.05), with preservation of neurofilament immunoreactive fibers in the epicardium of MPTP-treated monkeys. Alpha-synuclein deposits were observed in sections of the anterior left ventricle of MPTP-treated monkeys but not in control animals, whereas phosphorylated synuclein aggregates were not observed in either controls or MPTP-treated monkeys. Conclusion The peripheral autonomic system can also be affected by neurotoxins that specifically inhibit mitochondrial complex I.

Glial cell line-derived neurotrophic factor (GDNF) remains the most potent neurotrophic factor for dopamine neurons. Despite its potential as treatment for Parkinson's disease (PD), its clinical application has been hampered by safety and efficacy concerns associated with GDNF's short in vivo half-life and with significant brain delivery obstacles. Drug formulation systems such as microparticles (MPs) may overcome these issues providing protein protection from degradation and sustained drug release over time. We therefore sought to evaluate the efficacy and safety of GDNF delivered via injectable biodegradable MPs in a clinically relevant model of PD and to investigate the mechanism contributing to their beneficial effects. MPs were injected unilaterally into the putamen of parkinsonian monkeys with severe nigrostriatal degeneration. Notably, a single administration of the microencapsulated neurotrophic factor achieved sustained GDNF levels in the brain, providing motor improvement and dopaminergic function restoration. This was reflected by a bilateral increase in the density of striatal dopaminergic neurons 9 months after treatment. Moreover, GDNF was retrogradely transported to the substantia nigra increasing bilaterally the number of dopaminergic and total neurons, regardless of the severe degeneration. GDNF-MP injection within the putamen elicited no adverse effects such as immunogenicity, cerebellar degeneration or weight loss. MPs are therefore a safe, efficient vehicle for sustained protein delivery to the brain, supporting the therapeutic benefit of GDNF when encapsulated within MPs for brain repair. Overall, these findings constitute important groundwork for GDNF-MP clinical development.

Much controversy exists concerning the effect of levodopa on striatal dopaminergic markers in Parkinson's disease (PD) and its influence on functional neuroimaging. To deal with this issue we studied the impact of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and chronic levodopa administration on striatal (18)F-DOPA uptake (Ki) in an animal model of PD. The levels of several striatal dopaminergic markers and the number of surviving dopaminergic neurons in the substantia nigra (SN) were also assessed. Eleven Macaca fascicularis were included in the study. Eight animals received weekly intravenous injections of MPTP for 7weeks and 3 intact animals served as controls. MPTP-monkeys were divided in two groups. Group I was treated with placebo while Group II received levodopa. Both treatments were maintained for 11months and then followed by a washout period of 6months. (18)F-DOPA positron emission tomography (PET) scans were performed at baseline, after MPTP intoxication, following 11months of treatment, and after a washout period of 1, 3 and 6months. Monkeys were sacrificed 6months after concluding either placebo or levodopa treatment and immediately after the last (18)F-DOPA PET study. Striatal dopamine transporter (DAT) content, tyrosine hydroxylase (TH) content and aromatic l-amino acid decarboxylase (AADC) content were assessed. In Group II (18)F-DOPA PET studies performed at 3 and 6months after interrupting levodopa showed a significantly increased Ki in the anterior putamen as compared to Group I. Levodopa and placebo treated animals exhibited a similar number of surviving dopaminergic cells in the SN. Striatal DAT content was equally reduced in both groups of animals. Animals in Group I exhibited a significant decrease in TH protein content in all the striatal regions assessed. However, in Group II, TH levels were significantly reduced only in the anterior and posterior putamen. Surprisingly, in the levodopa-treated animals the TH levels in the posterior putamen were significantly lower than those in the placebo group. AADC levels in MPTP groups were similar to those of control animals in all striatal areas analyzed. This study shows that chronic levodopa administration to monkeys with partial nigrostriatal degeneration followed by a washout period induces modifications in the functional activity of the dopaminergic nigrostriatal pathway.

Department of Industry of the Basque Government [S-PE12UN030]; Spanish Health Ministry [FIS PI08/1866, FIS PI13/01250]

UTE-project/Foundation for Applied Medical Research (FIMA)