Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. 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

Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the “hydrophobic motif” of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells

The Return of Results in Genetic Testing: Who Owes What to Whom, When, and Why?

The field of genetic research has revolutionized modern medicine and will continue to do so in the years to come. For the people whose biological materials form the basis for this research, however, the research process may also lead to personal discoveries—namely, it may expose information about their health, genetic predispositions, and other gene-linked characteristics. Researchers who uncover this kind of personal genetic information are likewise confronted with the question of whether they should—or must—provide their subjects with feedback about their results.For subjects and researchers alike, the answer is unclear. Presently, there is little guidance as to these parties’ rights and responsibilities when it comes to the return of genetic results in a research setting. As a result, neither party has a clearly defined understanding of what to expect from the research relationship. This Article draws on recognized ethical and legal foundations to propose that genetic researchers should owe three limited legal duties to their research subjects regarding planning for, acquiring informed consent about, and reporting certain genetic findings. Considering the wide variation among individuals in terms of what genetic information they would like to know, this Article balances concerns for individual autonomy with the right to acquire personal health information, and it weighs those interests against the potential cost to socially beneficial genetic research. In balancing these considerations, this Article’s proposals for a limited set of duties offer a careful step toward clearly defining the rights and responsibilities of genetic researchers and their subjects

Changes in Caregiving Status and Intensity and Sleep Characteristics Among High and Low Stressed Older Women.

STUDY OBJECTIVES:To examine whether change in caregiving status and intensity among community-dwelling older women was associated with sleep characteristics at follow-up, and whether perceived stress modified these associations. METHODS:The sample included 800 women aged 65 years or older who completed baseline and second follow-up interviews in the Caregiver-Study of Osteoporotic Fractures (Caregiver-SOF). Respondents were categorized into four groups based on change in caregiving status and intensity between the two time points: continuous noncaregivers, ceased caregivers, low-intensity caregivers (continuous caregivers with low/decreased intensity), and high-intensity caregivers (continuous caregivers with high/increased intensity or new caregivers). Perceived Stress Scale scores at the second follow-up were dichotomized into high versus low stress. Sleep outcomes at SOF Visit 8 (which overlapped with Caregiver-SOF second follow-up) included the Pittsburgh Sleep Quality Index total score; and actigraphy-measured total sleep time, sleep efficiency, wake after sleep onset, and sleep latency. RESULTS:Multivariate-adjusted sleep characteristics did not differ significantly across caregiving groups. Among high-intensity caregivers, however, those with high stress levels had significantly longer wake after sleep onset (mean 82.3 minutes, 95% confidence interval = 70.9-93.7) than those with low stress levels (mean 65.4 minutes, 95% confidence interval = 55.2-75.7). No other sleep outcomes were modified by stress levels. Further, higher stress was significantly associated with worse Pittsburgh Sleep Quality Index scores, regardless of the caregiving group. CONCLUSIONS:Overall, sleep characteristics did not differ among noncaregivers, ceased caregivers, or those with high-/low-intensity caregiving among older women. However, subgroups of caregivers may be vulnerable to developing sleep problems, particularly those with high stress levels