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Mutations in theSNCAgene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons in the substantia nigra, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from theSNCAmutation during development, to its end stage pathology is unknown. Utilising human induced pluripotent stem cells (hiPSCs) withSNCAmutations, we resolved the temporal sequence of pathophysiological events that occur during neuronal differentiation in order to discover the early, and likely causative, events in synucleinopathies. We adapted a small molecule-based protocol that generates highly enriched midbrain dopaminergic (mDA) neurons (>80%). We characterised their molecular identity using single-cell RNA sequencing and their functional identity through the synthesis and secretion of dopamine, the ability to generate action potentials, and form functional synapses and networks. RNA velocity analyses confirmed the developmental transcriptomic trajectory of midbrain neural precursors into mDA neurons using our approach, and identified key driver genes in mDA neuronal development. To characterise the synucleinopathy, we adopted super-resolution methods to determine the number, size and structure of aggregates inSNCA-mutant mDA neurons. At one week of differentiation, prior to maturation to mDA neurons of molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, inSNCA-mutant midbrain immature neurons. The aggregation progresses over time to accumulate phosphorylated aggregates, and later fibrillar aggregates. When the midbrain neurons were functional, we observed evidence of impaired physiological calcium signalling, with raised basal calcium, and impairments in cytosolic and mitochondrial calcium efflux. Once midbrain identity fully developed,SNCA-mutant neurons exhibited bioenergetic impairments, mitochondrial dysfunction and oxidative stress. During the maturation of mDA neurons, upregulation of mitophagy and autophagy occured, and ultimately these multiple cellular stresses lead to an increase in cell death by six weeks post-differentiation. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

Aggregation of α-Synuclein (α-Syn) drives Parkinson’s disease, although the initial stages of self-assembly and structural conversion have not been captured inside neurons. We track the intracellular conformational states of α-Syn utilizing a single-molecule FRET biosensor, and show that α-Syn converts from its monomeric state to form two distinct oligomeric states in neurons in a concentration dependent, and sequence specific manner. 3D FRET-CLEM reveals the structural organization, and location of aggregation hotspots inside the cell. Notably multiple intracellular seeding events occur preferentially on membrane surfaces, especially mitochondrial membranes. The mitochondrial lipid, cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial ROS generation, which accelerates the oligomerization of A53T α-Syn, and ultimately causes permeabilization of mitochondrial membranes, and cell death. Patient iPSC derived neurons harboring A53T mutations exhibit accelerated oligomerization that is dependent on mitochondrial ROS, early mitochondrial permeabilization and neuronal death. Our study highlights a mechanism of de novo oligomerization at the mitochondria and its induction of neuronal toxicity.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

ImportanceType 2 diabetes (T2DM) is an established risk factor for developing Parkinson’s disease (PD) but its effect on disease progression is not well understood.ObjectiveTo examine the effects of co-morbid T2DM on Parkinson’s disease progression and quality of life.DesignWe analysed data from the Tracking Parkinson’s study, a large multi-centre prospective study in the UK.ParticipantsThe study included 1930 adults with recent onset PD, recruited between February 2012 and May 2014, and followed up regularly thereafter.ExposureA diagnosis of pre-existing T2DM was based on self-report at baseline. After controlling for confounders, an evaluation of how T2DM affects PD was performed by comparing symptom severity scores; and analyses using multivariable mixed models was used to determine the effects of T2DM on Parkinson’s disease progression.Main Outcomes and MeasuresThe impact of T2DM on Parkinson’s disease severity was derived from scores collected using the Movement Disorders Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS), Non-Motor Symptoms Scale (NMSS), Montreal Cognitive Assessment (MoCA), Questionnaire for impulsive-compulsive disorders in PD (QUIP), Leeds Anxiety and Depression Scale (LADS), and Schwab and England ADL scale.ResultsWe identified 167 (8.7%) patients with PD and T2DM (PD+T2DM) and 1763 (91.3%) with PD without T2DM (PD). Patients with T2DM had more severe motor symptoms, as assessed by MDS-UPDS III 25.8 (0.9) vs 22.5 (0.3) p=0.002, had significantly faster motor symptom progression over time (p=0.012), and T2DM was an independent predictor for the development of substantial gait impairment (HR 1.55, CI 1.07-2.23, p=0.020). Patients were more likely to have loss of independence (OR 2.08, CI 1.34-3.25, p=0.001); and depression (OR 1.62, CI 1.10-2.39, p=0.015), and developed worsening mood (p=0.041) over time compared to the PD group. T2DM was also an independent predictor for the development mild cognitive impairment (HR 1.7, CI 1.24-2.51, p=0.002) over timeConclusions and relevanceT2DM is associated with faster disease progression in PD, highlighting an interaction between these two diseases. As it is a potentially modifiable, metabolic state, with multiple peripheral and central targets for intervention, it may represent a target for ameliorating parkinsonian symptoms, and progression to disability and dementia.Key pointsQuestionWhat is the impact of Type 2 diabetes on Parkinson’s disease progression?FindingsIn this prospective study of 1930 patients with recent onset PD, T2DM is an independent risk factor for more severe motor features, non-motor symptoms, and poorer quality of life; and importantly is associated with faster motor and non-motor symptom progression, and increases the risk of developing cognitive impairment.MeaningT2DM is identified as a new factor that alters Parkinson’s disease progression. T2DM predicts both motor and non-motor symptom progression in PD and is associated with poorer quality of life, highlighting an interaction of two chronic disease states. This highlights a particular need for improved treatment in this subgroup of patients with Parkinson’s.

The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.