Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

A topographical atlas of α-synuclein dosage and cell type-specific expression in adult mouse brain and peripheral organs.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and presents pathologically with Lewy pathology and dopaminergic neurodegeneration. Lewy pathology contains aggregated α-synuclein (αSyn), a protein encoded by the SNCA gene which is also mutated or duplicated in a subset of familial PD cases. Due to its predominant presynaptic localization, immunostaining for the protein results in a diffuse reactivity pattern, providing little insight into the types of cells expressing αSyn. As a result, insight into αSyn expression-driven cellular vulnerability has been difficult to ascertain. Using a combination of knock-in mice that target αSyn to the nucleus (SncaNLS) and in situ hybridization of Snca in wild-type mice, we systematically mapped the topography and cell types expressing αSyn in the mouse brain, spinal cord, retina, and gut. We find a high degree of correlation between αSyn protein and RNA levels and further identify cell types with low and high αSyn content. We also find high αSyn expression in neurons, particularly those involved in PD, and to a lower extent in non-neuronal cell types, notably those of oligodendrocyte lineage, which are relevant to multiple system atrophy pathogenesis. Surprisingly, we also found that αSyn is relatively absent from select neuron types, e.g., ChAT-positive motor neurons, whereas enteric neurons universally express some degree of αSyn. Together, this integrated atlas provides insight into the cellular topography of αSyn, and provides a quantitative map to test hypotheses about the role of αSyn in network vulnerability, and thus serves investigations into PD pathogenesis and other α-synucleinopathies.

Characterizing the differential distribution and targets of Sumo1 and Sumo2 in the mouse brain.

SUMOylation is an evolutionarily conserved eukaryotic posttranslational protein modification with broad biological relevance. Differentiating between the major small ubiquitin-like modifier (SUMO) paralogs and uncovering paralog-specific functions in vivo has long been very difficult. To overcome this problem, we generated His6-HA-Sumo2 and HA-Sumo2 knockin mouse lines, expanding upon our existing His6-HA-Sumo1 mouse line, to establish a "toolbox" for Sumo1-Sumo2 comparisons in vivo. Leveraging the specificity of the HA epitope, we performed whole-brain imaging and uncovered regional differences between Sumo1 and Sumo2 expression. At the subcellular level, Sumo2 was specifically detected in extranuclear compartments, including synapses. Immunoprecipitation coupled with mass spectrometry identified shared and specific neuronal targets of Sumo1 and Sumo2. Target validation using proximity ligation assays provided further insight into the subcellular distribution of neuronal Sumo2-conjugates. The mouse models and associated datasets provide a powerful framework to determine the native SUMO "code" in cells of the central nervous system.

Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

BACKGROUND: Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction. RESULTS: Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1-/- and p35-/- neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1-/- neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination. CONCLUSIONS: Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.

Constitutive nuclear accumulation of endogenous alpha-synuclein in mice causes motor impairment and cortical dysfunction, independent of protein aggregation.

A growing body of evidence suggests that nuclear alpha-synuclein (αSyn) plays a role in the pathogenesis of Parkinson's disease (PD). However, this question has been difficult to address as controlling the localization of αSyn in experimental systems often requires protein overexpression, which affects its aggregation propensity. To overcome this, we engineered SncaNLS mice, which localize endogenous αSyn to the nucleus. We characterized these mice on a behavioral, histological and biochemical level to determine whether the increase of nuclear αSyn is sufficient to elicit PD-like phenotypes. SncaNLS mice exhibit age-dependent motor deficits and altered gastrointestinal function. We found that these phenotypes were not linked to αSyn aggregation or phosphorylation. Through histological analyses, we observed motor cortex atrophy in the absence of midbrain dopaminergic neurodegeneration. We sampled cortical proteomes of SncaNLS mice and controls to determine the molecular underpinnings of these pathologies. Interestingly, we found several dysregulated proteins involved in dopaminergic signaling, including Darpp32, Pde10a and Gng7, which we further confirmed was decreased in cortical samples of the SncaNLS mice compared with controls. These results suggest that chronic endogenous nuclear αSyn can elicit toxic phenotypes in mice, independent of its aggregation. This model raises key questions related to the mechanism of αSyn toxicity in PD and provides a new model to study an underappreciated aspect of PD pathogenesis.

Emotional and Mental Wellbeing Following COVID-19 Public Health Measures on People Living With Dementia and Carers.

BACKGROUND: To date, there appears to be no evidence on the longer-term impacts caused by COVID-19 and its related public health restrictions on some of the most vulnerable in our societies. The aim of this research was to explore the change in impact of COVID-19 public health measures on the mental wellbeing of people living with dementia (PLWD) and unpaid carers. METHOD: Semi-structured, follow-up telephone interviews were conducted with PLWD and unpaid carers between June and July 2020. Participants were asked about their experiences of accessing social support services during the pandemic, and the impact of restrictions on their daily lives. RESULTS: 20 interviews were conducted and thematically analyzed, which produced 3 primary themes concerning emotional responses and impact to mental health and wellbeing during the course of the pandemic: 1) Impact on mental health during lockdown, 2) Changes to mental health following easing of public health, and 3) The long-term effect of public health measures. CONCLUSIONS: The findings from this research shed light on the longer-term psychological impacts of the UK Government's public health measures on PLWD and their carers. The loss of social support services was key in impacting this cohort mentally and emotionally, displaying a need for better psychological support, for both carers and PLWD.

Navigating the new normal: accessing community and institutionalised care for dementia during COVID-19.

OBJECTIVES: Little is known about how community services and institutional care settings have adapted to providing support since the COVID-19 pandemic. The aim was to explore how these care services have adapted during the pandemic in the UK and are providing care to people living with dementia (PLWD) and carers. METHOD: Semi-structured telephone interviews were conducted in June and July 2020 with 16 purposefully sampled unpaid dementia carers. Participants were asked about their experiences of accessing care services since the lockdown, and whether they were beneficial, if accessed at all. RESULTS: Three themes were identified: (1) Impacts of no activities; (2) Difficulties accessing care during the pandemic; (3) Remote vs. face-to-face support. Loss of access to previously enjoyed activities and having had to shield for many PLWD is suggested to have led to severe physical and cognitive deteriorations, advancing the dementia. Where remote support was available, this was helpful to some, but did not replace the benefits of face-to-face support. Where PLWD were residing in a care home, carers had very limited remote access. CONCLUSIONS: This is the first study to explore the impact on carers both from a community and institutionalised care angle. Few care services have adapted to providing remote support. With the vaccine taking time to be accessible to everyone, it is vital for organisations to work closely with carers and PLWD to adapt services to provide much needed support.

Resilience and supporting people living with dementia during the time of COVID-19; A qualitative study.

OBJECTIVES: To explore the different factors of resilience for people living with dementia and unpaid carers, in response to sudden changes in care and lifestyle during the COVID-19 pandemic. METHODS: Unpaid carers and people living with dementia were offered telephone interviews in April 2020 to discuss their experiences since the COVID-19 pandemic. Participants were asked about the benefits and challenges of accessing dementia support, as well as coping, symptoms, strategies and impacts. Each transcript was analysed using inductive and deductive thematic analysis by two researchers. FINDINGS: Semi-structured interviews from 50 participants (n = 42 unpaid carers and n = 8 people living with dementia) reported protective and risk factors of resilience concerning (1) communication, (2) adaptations, (3) support networks and (4) lifestyle factors and coping mechanisms. CONCLUSIONS: Resilience factors considered both organisational factors for external support, along with individual coping mechanisms. Organisations and social support services should consider resilience factors in future service planning, to better support people living with dementia, or caring someone living with dementia, during times of great stress. The ecological model of resilience established from this research refers to resilience during times of unexpected change in the COVID-19 pandemic; however, it could be considered relevant in other periods of high stress within this cohort.

The potential dangers of not understanding COVID-19 public health restrictions in dementia: "It's a groundhog day - every single day she does not understand why she can't go out for a walk".

BACKGROUND: Sudden public health restrictions can be difficult to comprehend for people with cognitive deficits. However, these are even more important for them to adhere to due to their increased levels of vulnerability, particularly to COVID-19. With a lack of previous evidence, we explored the understanding and changes in adherence to COVID-19 public health restrictions over time in people living with dementia (PLWD). METHODS: Unpaid carers and PLWD were interviewed over the phone in April 2020, shortly after the nationwide UK lockdown, with a proportion followed up from 24th June to 10th July. Participants were recruited via social care and third sector organisations across the UK, and via social media. FINDINGS: A total of 70 interviews (50 baseline, 20 follow-up) were completed with unpaid carers and PLWD. Five themes emerged: Confusion and limited comprehension; Frustration and burden; Putting oneself in danger; Adherence to restrictions in wider society; (Un) changed perceptions. Most carers reported limited to no understanding of the public health measures in PLWD, causing distress and frustration for both the carer and the PLWD. Due to the lack of understanding, some PLWD put themselves in dangerous situations without adhering to the restrictions. PLWD with cognitive capacity who participated understood the measures and adhered to these. DISCUSSION: In light of the new second wave of the pandemic, public health measures need to be simpler for PLWD to avoid unwilful non-adherence. Society also needs to be more adaptive to the needs of people with cognitive disabilities more widely, as blanket rules cause distress to the lives of those affected by dementia.

"A piece of paper is not the same as having someone to talk to": accessing post-diagnostic dementia care before and since COVID-19 and associated inequalities.

BACKGROUND: Social support services such as day care centres are important in post-diagnostic dementia care to enable people living with dementia stay at home for longer. Little research has addressed potential inequalities in access, with no research on variations before and since COVID-19. The aim of this study was to explore inequalities in social support service usage before and since the pandemic. METHODS: Unpaid carers and people living with dementia were interviewed over the phone about their experiences of accessing social support services before and since the COVID-19 pandemic. Transcripts were analysed for key themes using inductive and deductive thematic analysis. RESULTS: Fifty participants (42 unpaid carers; eight people living with dementia) were interviewed, and five themes identified: (1) Service issues; (2) Access issues; (3) Relying on own initiative; (4) New inequalities due to COVID-19; and (5) Missing out on the benefits of support services. Participants reported transport, finances, and location as factors reducing their ability to access support service pre-COVID, with inequalities remaining and at times exacerbated since. Carers and people living with dementia also reported struggling with accessing basic necessities during COVID, including food and medicines. CONCLUSIONS: Considering the benefits of accessing support services, resourced procedures and facilities are needed to maintain access to support services with more accessible remote support provision, enabling people from all backgrounds to access the care they need.

Age-associated insolubility of parkin in human midbrain is linked to redox balance and sequestration of reactive dopamine metabolites.

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.

COVID-19-related social support service closures and mental well-being in older adults and those affected by dementia: a UK longitudinal survey.

BACKGROUND: The COVID-19 pandemic has had a major impact on delivery of social support services. This might be expected to particularly affect older adults and people living with dementia (PLWD), and to reduce their well-being. AIMS: To explore how social support service use by older adults, carers and PLWD, and their mental well-being changed over the first 3 months since the pandemic outbreak. METHODS: Unpaid dementia carers, PLWD and older adults took part in a longitudinal online or telephone survey collected between April and May 2020, and at two subsequent timepoints 6 and 12 weeks after baseline. Participants were asked about their social support service usage in a typical week prior to the pandemic (at baseline), and in the past week at each of the three timepoints. They also completed measures of levels of depression, anxiety and mental well-being. RESULTS: 377 participants had complete data at all three timepoints. Social support service usage dropped shortly after lockdown measures were imposed at timepoint 1 (T1), to then increase again by T3. The access to paid care was least affected by COVID-19. Cases of anxiety dropped significantly across the study period, while cases of depression rose. Well-being increased significantly for older adults and PLWD from T1 to T3. CONCLUSIONS: Access to social support services has been significantly affected by the pandemic, which is starting to recover slowly. With mental well-being differently affected across groups, support needs to be put in place to maintain better well-being across those vulnerable groups during the ongoing pandemic.

Impact of COVID-19 related social support service closures on people with dementia and unpaid carers: a qualitative study.

OBJECTIVES: Accessing social care and social support services is key to support the well-being of people living with dementia (PLWD) and unpaid carers. COVID-19 has caused sudden closures or radical modifications of these services, and is resulting in prolonged self-isolation. The aim of this study was to explore the effects of COVID-19 related social care and support service changes and closures on the lives of PLWD and unpaid carers. METHOD: PLWD and unpaid carers were interviewed via telephone in April 2020. Transcripts were analysed using thematic analysis. Demographic characteristics including household Index of Multiple Deprivation score and weekly hours of social support service usage before and since the COVID-19 outbreak were also collected. Paired samples t-tests was used to compare the mean of weekly hours of social support service usage before and since the outbreak. RESULTS: 50 semi-structured interviews were conducted with unpaid carers (n = 42) and PLWD (n = 8). There was a significant reduction in social support service usage since the outbreak. Thematic analysis identified three overarching themes: (1) Loss of control; (2) Uncertainty; (3) Adapting and having to adapt to the new normal. Carers and PLWD were greatly affected by the sudden removal of social support services, and concerned about when services would re-open. Carers were worried about whether the person they cared for would still be able to re-join social support services. CONCLUSIONS: PLWD and carers need to receive specific practical and psychological support during the pandemic to support their well-being, which is severely affected by public health restrictions.

A UK survey of COVID-19 related social support closures and their effects on older people, people with dementia, and carers.

OBJECTIVES: The aim of this national survey was to explore the impact of COVID-19 public health measures on access to social support services and the effects of closures of services on the mental well-being of older people and those affected by dementia. METHODS: A UK-wide online and telephone survey was conducted with older adults, people with dementia, and carers between April and May 2020. The survey captured demographic and postcode data, social support service usage before and after COVID-19 public health measures, current quality of life, depression, and anxiety. Multiple linear regression analysis was used to explore the relationship between social support service variations and anxiety and well-being. RESULTS: Five hundred and sixty-nine participants completed the survey (61 people with dementia, 285 unpaid carers, and 223 older adults). Paired samples t-tests and X2 -tests showed that the mean hour of weekly social support service usage and the number of people having accessed various services was significantly reduced post COVID-19. Multiple regression analyses showed that higher variations in social support service hours significantly predicted increased levels of anxiety in people with dementia and older adults, and lower levels of mental well-being in unpaid carers and older adults. CONCLUSIONS: Being unable to access social support services due to COVID contributed to worse quality of life and anxiety in those affected by dementia and older adults across the UK. Social support services need to be enabled to continue providing support in adapted formats, especially in light of continued public health restrictions for the foreseeable future.

Decision-making for receiving paid home care for dementia in the time of COVID-19: a qualitative study.

BACKGROUND: The lockdown imposed in the UK on the 23rd of March and associated public health measures of social distancing are likely to have had a great impact on care provision. The aim of this study was to explore the decision-making processes of continued paid home care support for dementia in the time of COVID-19. METHODS: Unpaid carers caring for a person living with dementia (PLWD) who were accessing paid home care before COVID-19 and residing in the UK were eligible to take part. Participants were interviewed over the phone and asked about their experiences of using paid home care services before and since COVID-19, and their decision-making processes of accessing paid home care since the outbreak and public health restrictions. RESULTS: Fifteen unpaid carers, who were also accessing paid care support for the PLWD before COVID-19, were included in the analysis. Thematic analysis identified three overarching themes: (1) Risk; (2) Making difficult choices and risk management; and (3) Implications for unpaid carers. Many unpaid carers decided to discontinue paid carers entering the home due to the risk of infection, resulting in unpaid carers having to pick up the care hours to support the person living with dementia. CONCLUSIONS: This is the first study to report on the impact of COVID-19 on paid home care changes in dementia. Findings raise implications for providing better Personal Protective Equipment for paid carers, and to support unpaid carers better in their roles, with the pandemic likely to stay in place for the foreseeable future.

The pro-death role of Cited2 in stroke is regulated by E2F1/4 transcription factors.

We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.

Pink1 regulates FKBP5 interaction with AKT/PHLPP and protects neurons from neurotoxin stress induced by MPP.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

DJ-1 modulates the unfolded protein response and cell death via upregulation of ATF4 following ER stress.

The unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress is a feature of many neurodegenerative diseases including Alzheimer's disease, Huntington's disease and Parkinson's disease (PD). Although the vast majority of PD is sporadic, mutations in a number of genes including PARK7 which encodes the protein DJ-1 have been linked to early-onset, familial PD. In this regard, both PD of sporadic and genetic origins exhibit markers of ER stress-induced UPR. However, the relationship between pathogenic mutations in PARK7 and ER stress-induced UPR in PD pathogenesis remains unclear. In most contexts, DJ-1 has been shown to protect against neuronal injury. However, we find that DJ-1 deficiency ameliorates death in the context of acute ER stress in vitro and in vivo. DJ-1 loss decreases protein and transcript levels of ATF4, a transcription factor critical to the ER response and reduces the levels of CHOP and BiP, its downstream effectors. The converse is observed with DJ-1 over-expression. Importantly, we find that over-expression of wild-type and PD-associated mutant form of PARK7L166P, enhances ER stress-induced neuronal death by regulating ATF4 transcription and translation. Our results demonstrate a previously unreported role for wild-type and mutant DJ-1 in the regulation of UPR and provides a potential link to PD pathogenesis.

Regulation of myeloid cell phagocytosis by LRRK2 via WAVE2 complex stabilization is altered in Parkinson's disease.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in both familial and sporadic Parkinson's disease (PD), yet its pathogenic role remains unclear. A previous screen in Drosophila identified Scar/WAVE (Wiskott-Aldrich syndrome protein-family verproline) proteins as potential genetic interactors of LRRK2 Here, we provide evidence that LRRK2 modulates the phagocytic response of myeloid cells via specific modulation of the actin-cytoskeletal regulator, WAVE2. We demonstrate that macrophages and microglia from LRRK2-G2019S PD patients and mice display a WAVE2-mediated increase in phagocytic response, respectively. Lrrk2 loss results in the opposite effect. LRRK2 binds and phosphorylates Wave2 at Thr470, stabilizing and preventing its proteasomal degradation. Finally, we show that Wave2 also mediates Lrrk2-G2019S-induced dopaminergic neuronal death in both macrophage-midbrain cocultures and in vivo. Taken together, a LRRK2-WAVE2 pathway, which modulates the phagocytic response in mice and human leukocytes, may define an important role for altered immune function in PD.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.