The natural history study of preclinical genetic Creutzfeldt-Jakob Disease (CJD): a prospective longitudinal study protocol.

BACKGROUND: Creutzfeldt-Jakob Disease (CJD) is the most common prion disease in humans causing a rapidly progressive neurological decline and dementia and is invariably fatal. The familial forms (genetic CJD, gCJD) are caused by mutations in the PRNP gene encoding for the prion protein (PrP). In Israel, there is a large cluster of gCJD cases, carriers of an E200K mutation in the PRNP gene, and therefore the largest population of at-risk individuals in the world. The mutation is not necessarily sufficient for the formation and accumulation of the pathological prion protein (PrPsc), suggesting that other, genetic and non-genetic factors affect the age at symptoms onset. Here we present the protocol of a cross-sectional and longitudinal natural history study of gCJD patients and first-degree relatives of gCJD patients, aiming to identify biological markers of preclinical CJD and risk factors for phenoconversion. METHODS: The study has two groups: Patients diagnosed with gCJD, and first-degree healthy relatives (HR) (both carriers and non-carriers of the E200K mutation in the PRNP gene) of patients diagnosed with gCJD. At baseline, and at the end of every year, healthy participants are invited for an "in-depth" visit, which includes a clinical evaluation, blood and urine collection, gait assessment, brain MRI, lumbar puncture (LP), and Polysomnography (PSG). At 6 months from baseline, and then halfway through each year, participants are invited for a "brief" visit, which includes a clinical evaluation, short cognitive assessment, and blood and urine collection. gCJD patients will be invited for one "in-depth" visit, similar to the baseline visit of healthy relatives. DISCUSSION: This continuous follow-up of the participants and the frequent assessments will allow early identification and diagnosis in case of conversion into disease. The knowledge generated from this study is likely to advance the understanding of the underlying clinicopathological processes that occur at the very beginning of CJD, as well as potential genetic and environmental risk factors for the development of the disease, therefore advancing the development of safe and efficient interventions. TRIAL REGISTRATION: The study is an observational study. It has registered retrospectively in https://clinicaltrials.gov/ and has been assigned an identification number NCT05746715.

The GBA-370Rec Parkinson's disease risk haplotype harbors a potentially pathogenic variant in the mitochondrial gene SLC25A44.

GBA variations are common risk factors for Parkinson's disease (PD), and are found in 21.7% of Ashkenazi PD patients (AJ-PD), 4.23% of them carry an allele, 370Rec, which is different from the common GBA-N370S allele. Using whole-genome-sequencing of 370Rec carriers, N370S carriers, and non-carriers, we characterize the unique 370Rec haplotype in AJ-PDs, and show that it harbors a missense variant replacing the highly conserved methionine-27 with valine in the transmembrane domain of the mitochondrial SLC25A44.

[18F] FDOPA PET may confirm the clinical diagnosis of Parkinson's disease by imaging the nigro-striatal pathway and the sympathetic cardiac innervation: Proof-of-concept study.

Autonomic involvement, including cardiac denervation, may precede the motor symptoms of Parkinson's disease by several years. L-3,4-dihydroxy-6-[18F] fluoro-phenylalanine is a positron emitter and a true analog of L-dopa, used in clinical practice to assess striatal dopaminergic integrity. The present study aimed to assess the feasibility of evaluating cardiac sympathetic denervation in Parkinson's disease patients using L-3,4-dihydroxy-6-[18F] fluoro-phenylalanine positron emission tomography/computed tomography. Patients referred for an L-3,4-dihydroxy-6-[18F] fluoro-phenylalanine positron emission tomography/computed-tomography between July 2015 and May 2017 to evaluate striatal presynaptic dopaminergic integrity underwent a heart positron emission tomography scan following a brain positron emission tomography scan. L-3,4-dihydroxy-6-[18F] fluoro-phenylalanine uptake in the left ventricle was quantified using CarimasT⁢M software and compared between patients with and without Parkinson's disease. The area under the receiver operating characteristic curve was used to evaluate the ability of the left ventricular mean standardized uptake value to discriminate between patients with Parkinson's disease and those with other extrapyramidal syndromes. Seventy-six patients were included, of whom 52 were diagnosed with Parkinson's disease. The mean L-3,4-dihydroxy-6-[18F] fluoro-phenylalanine left ventricular mean standardized uptake value was lower in the Parkinson's disease patients compared to the non- Parkinson's disease patients (1.08 ± 0.21 vs. 1.24 ± 0.32, P = 0.015). The left ventricular mean standardized uptake value was able to discriminate between Parkinson's disease and non- Parkinson's disease patients (the area under the receiver operating characteristic curve = 0.641, P = 0.049). In conclusion, quantification of cardiac L-3,4-dihydroxy-6-[18F] fluoro-phenylalanine uptake may be able to differentiate between patients with and without Parkinson's disease. Validation of this finding in more substantial, prospective trials are warranted.

Revisiting the non-Gaucher-GBA-E326K carrier state: Is it sufficient to increase Parkinson's disease risk?

BACKGROUND: GBA variants are the most common genetic risk factors for Parkinson's disease (PD) world-wide, and can be found in up to 20% of Ashkenazi PD patients. The E326K variant, which is not considered a Gaucher's disease causing mutation, was recently shown to increase the risk for PD. Since E326K is a common variant among Europeans, Finnish and Ashkenazi (2.4, 8.6 and 1.2% carrier rate, respectively), we aimed to refine its involvement in PD. METHODS: 1200 consecutively recruited PD patients of a full Ashkenazi origin were genotyped for 10 GBA variants, the LRRK2-G2019S and the SMPD1-L302P. Alleles' frequencies were compared to controls, composed of 378 elderly healthy individuals and the non-neuro gnomAD Ashkenazi database. Odds-Ratio (OR) and age-at-motor-symptom-onset (AAO) were also calculated for all genotypes. RESULTS: All allelic variations tested had significant allelic ORs, demonstrating a wide range (1.86-12.84). The lowest allelic OR was observed for E326K (p = .013). Forty-five patients (of 1200, 3.75%) had at least two mutations (of the 12 tested), compared to 2 (0.53%) among 378 controls (p = .0013). Of the E326K carrier patients, 37% (10/27) carried additional mutations and the genotypic OR for individuals who carried only the E326K variant was 1.07. It did not reach statistical significance even when simulating the expected carrier frequency of E326K in 100,000 Ashkenazi controls (p = .39). In addition, an additive effect was demonstrated for risk in carriers of two mutations, the LRRK2-G2019S and a mild-GBA mutation (N370S or R496H), compared to carriers of only one mutation in one of these genes (simulated OR 11.79 compared to 7.58 and 2.49, respectively). An additive effect was also suggested for earlier AAO (5.0 years earlier than in non-carriers, compared to 3.1 and 2.2 years, respectively). CONCLUSIONS: Compared to previous studies, we demonstrate here a higher frequency of PD patients that carry two mutations. The GBA-E326K is more likely to affect PD risk when accompanied by another mutation, and an additive effect on risk and earlier AAO was proposed for carriers of LRRK2/mild-GBA double mutations. Altogether, these data support an oligogenic approach to PD genetics.

Distinguishing Dementia With Lewy Bodies From Alzheimer Disease: What is the Influence of the GBA Genotype in Ashkenazi Jews?

Cognitive deficits beyond memory impairment, such as those affecting language production or executive functioning, can be useful in clinically distinguishing between dementia syndromes. We tested the hypothesis that Ashkenazi Jewish (AJ) patients who have dementia with Lewy bodies (DLB) and carry glucocerebrosidase (GBA) mutations will have verbal fluency deficits different from those found in Alzheimer disease (AD), whereas AJ patients with DLB who have no GBA mutations will have similar deficits in verbal fluency to those found in AD. We compared performance in phonemic and semantic verbal fluency tasks in 44 AJ patients with DLB and 20 patients with AD, matched for age, education, and age of immigration. All groups were found to have a deficit in semantic verbal fluency. On conducting the phonemic task, patients with DLB who carried GBA mutations scored more poorly than patients with AD, whereas DLB-noncarriers performed similarly to patients with AD. We suggest that verbal fluency tasks could serve as a possible clinical marker to subtype patients with DLB, with phonemic fluency being a marker for GBA-associated DLB.

Dissociation in awareness of memory and language decline in Alzheimer's disease.

OBJECTIVES: The aim of this study was to examine awareness of decline in memory and in language in individuals with Alzheimer's disease (AD), by comparing participant and informant ratings, as well as these ratings and actual test performance. METHODS: We analyzed data from 149 individuals with AD enrolled in the Alzheimer's Disease Neuroimaging Initiative (ADNI) who filled the Everyday Cognition questionnaire and performed memory and language tasks. RESULTS: Participants provided significantly lower assessments of decline than did informants for both memory and language. There was a negative association between informant ratings and memory test scores but no association between participant ratings and memory test scores. Both participant and informant ratings correlated negatively with performance on the language tests. Informant, but not participant, ratings contributed to the prediction of one memory variable beyond demographic factors. Participant ratings contributed to the prediction of language scores beyond demographic factors more than did informant ratings. CONCLUSIONS: The findings reflect better awareness of decline in language than of decline in memory in individuals with AD.

Cerebral Imaging Markers of GBA and LRRK2 Related Parkinson's Disease and Their First-Degree Unaffected Relatives.

Cerebral atrophy has been detected in patients with Parkinson's disease (PD) both with and without dementia, however differentiation based on genetic status has thus far not yielded robust findings. We assessed cortical thickness and subcortical volumes in a cohort of PD patients and healthy controls carriers of the G2019S mutation in the LRRK2 gene and the common GBA mutations, in an attempt to determine whether genetic status influences structural indexes. Cortical thickness and subcortical volumes were computed and compared between six groups of participants; idiopathic PD, GBA-PD, LRRK2-PD, non-manifesting non-carriers (NMNC), GBA-non-manifesting carriers (NMC) and LRRK2-NMC utilizing the FreeSurfer software program. All participants were cognitively intact based on a computerized cognitive assessment battery. Fifty-seven idiopathic PD patients, 9 LRRK2-PD, 12 GBA-PD, 49 NMNC, 41 LRRK2-NMC and 14 GBA-NMC participated in this study. Lower volumes among patients with PD compared to unaffected participants were detected in bilateral hippocampus, nucleus accumbens, caudate, thalamus, putamen and amygdala and the right pallidum (p = 0.016). PD patients demonstrated lower cortical thickness indexes in a majority of regions assessed compared with non-manifesting participants. No differences in cortical thickness and subcortical volumes were detected within each of the groups of participants based on genetic status. Mutations in the GBA and LRRK2 genes are not important determinants of cortical thickness and subcortical volumes in both patients with PD and non-manifesting participants. PD is associated with a general reduction in cortical thickness and sub-cortical atrophy even in cognitively intact patients.

Long-term cognitive outcomes in Susac syndrome: A case series.

Susac syndrome (SuS) presents with encephalopathy, visual disturbances, and hearing loss from immune-mediated microvascular occlusion. While acute SuS is well-described, long-term cognitive outcomes with current treatments are underknown. We assessed ten SuS patients treated in accordance with evidence-based guidelines using immunotherapies targeting humoral and cell-mediated pathways. Patients were followed for a median 3.6 years. Initially, cognition inversely correlated with corpus callosum lesions on MRI. All reported cognitive improvement; 5/10 patients had residual deficits in visual attention and executive function. Early, aggressive treatment was associated with good outcomes; extensive early corpus callosum lesions may identify patients at-risk of persistent cognitive deficits.

Dopamine, affordance and active inference.

The role of dopamine in behaviour and decision-making is often cast in terms of reinforcement learning and optimal decision theory. Here, we present an alternative view that frames the physiology of dopamine in terms of Bayes-optimal behaviour. In this account, dopamine controls the precision or salience of (external or internal) cues that engender action. In other words, dopamine balances bottom-up sensory information and top-down prior beliefs when making hierarchical inferences (predictions) about cues that have affordance. In this paper, we focus on the consequences of changing tonic levels of dopamine firing using simulations of cued sequential movements. Crucially, the predictions driving movements are based upon a hierarchical generative model that infers the context in which movements are made. This means that we can confuse agents by changing the context (order) in which cues are presented. These simulations provide a (Bayes-optimal) model of contextual uncertainty and set switching that can be quantified in terms of behavioural and electrophysiological responses. Furthermore, one can simulate dopaminergic lesions (by changing the precision of prediction errors) to produce pathological behaviours that are reminiscent of those seen in neurological disorders such as Parkinson's disease. We use these simulations to demonstrate how a single functional role for dopamine at the synaptic level can manifest in different ways at the behavioural level.

Dopamine and performance in a reinforcement learning task: evidence from Parkinson's disease.

The role dopamine plays in decision-making has important theoretical, empirical and clinical implications. Here, we examined its precise contribution by exploiting the lesion deficit model afforded by Parkinson's disease. We studied patients in a two-stage reinforcement learning task, while they were ON and OFF dopamine replacement medication. Contrary to expectation, we found that dopaminergic drug state (ON or OFF) did not impact learning. Instead, the critical factor was drug state during the performance phase, with patients ON medication choosing correctly significantly more frequently than those OFF medication. This effect was independent of drug state during initial learning and appears to reflect a facilitation of generalization for learnt information. This inference is bolstered by our observation that neural activity in nucleus accumbens and ventromedial prefrontal cortex, measured during simultaneously acquired functional magnetic resonance imaging, represented learnt stimulus values during performance. This effect was expressed solely during the ON state with activity in these regions correlating with better performance. Our data indicate that dopamine modulation of nucleus accumbens and ventromedial prefrontal cortex exerts a specific effect on choice behaviour distinct from pure learning. The findings are in keeping with the substantial other evidence that certain aspects of learning are unaffected by dopamine lesions or depletion, and that dopamine plays a key role in performance that may be distinct from its role in learning.

Decreased aperiodic neural activity in Parkinson's disease and dementia with Lewy bodies.

Neural oscillations and signal complexity have been widely studied in neurodegenerative diseases, whereas aperiodic activity has not been explored yet in those disorders. Here, we assessed whether the study of aperiodic activity brings new insights relating to disease as compared to the conventional spectral and complexity analyses. Eyes-closed resting-state electroencephalography (EEG) was recorded in 21 patients with dementia with Lewy bodies (DLB), 28 patients with Parkinson's disease (PD), 27 patients with mild cognitive impairment (MCI) and 22 age-matched healthy controls. Spectral power was differentiated into its oscillatory and aperiodic components using the Irregularly Resampled Auto-Spectral Analysis. Signal complexity was explored using the Lempel-Ziv algorithm (LZC). We found that DLB patients showed steeper slopes of the aperiodic power component with large effect sizes compared to the controls and MCI and with a moderate effect size compared to PD. PD patients showed steeper slopes with a moderate effect size compared to controls and MCI. Oscillatory power and LZC differentiated only between DLB and other study groups and were not sensitive enough to detect differences between PD, MCI, and controls. In conclusion, both DLB and PD are characterized by alterations in aperiodic dynamics, which are more sensitive in detecting disease-related neural changes than the traditional spectral and complexity analyses. Our findings suggest that steeper aperiodic slopes may serve as a marker of network dysfunction in DLB and PD features.

Differentiating dementia with Lewy bodies from Alzheimer's disease and Parkinson's disease dementia: an update on imaging modalities.

Dementia with Lewy bodies is the second most common cause of neurodegenerative dementia after Alzheimer's disease. Dementia with Lewy bodies can provide a diagnostic challenge due to the frequent overlap of clinical signs with other neurodegenerative conditions, namely Parkinson's disease dementia, and Alzheimer's disease. Part of this clinical overlap is due to the neuropathological overlap. Dementia with Lewy bodies is characterized by the accumulation of aggregated α-synuclein protein in Lewy bodies, similar to Parkinson's disease and Parkinson's disease dementia. However, it is also frequently accompanied by aggregation of amyloid-beta and tau, the pathological hallmarks of Alzheimer's disease. Neuroimaging is central to the diagnostic process. This review is an overview of both established and evolving imaging methods that can improve diagnostic accuracy and improve management of this disorder.

On the importance of using local tests and local norms in the assessment of memory.

The current study compared the assessment of memory with a translated story recall test and its original published norms and an equivalent local test with local norms. Analyses used data from 232 individuals with memory complaints who underwent neuropsychological evaluation at an outpatient memory clinic. One group of participants completed a translated test (N = 126) and another group completed a local test (N = 106). Additionally, participants completed tasks of word list recall, picture naming, and verbal fluency, all having local norms. The results showed that raw scores on the delayed story recall test, and on all other cognitive tasks, did not differ across groups, and the cross-task correlations were significant and similar in size in both groups. Yet, there was an interaction between group and standardized tests scores, whereby the standardized scores on the translated story recall test were equivalent to population mean, whereas all other scores fell below the mean. Conversion of raw scores to the original norms indicated that the performance of individuals with memory complaints was intact, while conversion of scores on a local test to local norms revealed the expected memory impairment. The findings highlight the importance of using local tests and local norms in the assessment of memory.

Differences in MS clinical and epidemiological characteristics between Ashkenazi and non-Ashkenazi Jewish patients in Israel: a retrospective single center study.

The prevalence and severity of Multiple Sclerosis (MS) varies across different ethnicities, with a tendency to a more severe phenotype in non-Caucasian populations.  Our objective was to evaluate the differences in disease phenotype between Ashkenazi Jewish and Non-Ashkenazi Jewish patients in Israel. We conducted a single center retrospective cohort study in which subjects were assigned to Ashkenazi or Non-Ashkenazi groups according to self-reported ancestry and disease severity was assessed using the expanded disability status (EDSS), MS severity score (MSSS), progression index (PI) and MRI metrics. 330 Ashkenazi Jewish (AJ) and 207 Non-Ashkenazi Jewish patients (Non-AJ) were included. Non-AJ had a younger age of disease onset (32.7 years vs. 35.7 years, p = 0.05), with a lower proportion of females (62.3% vs. 73.3%, p = 0.01). These differences were maintained within the subgroup of Israeli native patients. Ethnicity was a significant predictor of MSSS (ß = 0.601, p = 0.003), with a higher estimate than that of other epidemiological factors. To conclude, Non-AJ patients had an earlier age of onset and a more disabling disease as well as having a more balanced female to male ratio compared to AJ patients. These findings demonstrate variability of disease phenotype within Caucasian patient's dependent on their ethnicity despite equivalent access to healthcare services.

Event-related oscillations differentiate between cognitive, motor and visual impairments.

BACKGROUND: Parkinson's disease (PD) and dementia with Lewy bodies (DLB) share pathological and clinical similarities while differing in the timing and severity of motor cognitive and visual impairment. Previous EEG studies found abnormal neural oscillations in PD, mild cognitive impairment (MCI) and Alzheimer's disease, however, the electrophysiological signature of clinical symptoms is still unclear. We assessed the specificity of event-related oscillations in distinguishing between cognitive, motor and visual involvement in patients with neurodegenerative conditions. METHODS: EEG was recorded during a visual oddball task in 30 PD, 28 DLB, 30 MCI patients and 32 age-matched healthy controls. Target and non-target event-related power were examined in the time-frequency domain using complex Morlet wavelet convolution and compared within and between the study groups. RESULTS: MCI (z = - 1.8, p = 0.04, Cohen's d = - 0.5) and DLB (z = - 3.1, p < 0.001, d = - 1.0) patients showed decreased delta-band target event-related synchronization compared to participants with normal cognition. PD (z = 1.6, p = 0.05, d = 0.5) and DLB (z = 2.7, p < 0.01, d = 0.9) patients showed decreased beta suppression compared to MCI patients and controls. DLB patients with visual hallucinations (VH) showed decreased early-alpha suppression (z = 2.08, p = 0.019, d = 3.19, AUC = 1.0 ± 0.0) compared to DLB-VH-. CONCLUSIONS: Decreased event-related delta-band synchronization, reflecting a decline in information processing ability, was characteristic of cognitive impairment due to any cause. Decreased event-related beta suppression, reflecting impaired execution of motor action, was specific to PD and DLB. Decreased event-related early-alpha suppression was characteristic of the presence of VH in DLB. These findings show that specific oscillations may reflect specific clinical symptoms, being a marker of network dysfunction.

Decreased delta-band event-related power in dementia with Lewy bodies with a mutation in the glucocerebrosidase gene.

OBJECTIVE: To compare event-related oscillations in patients with dementia with Lewy bodies (DLB) who are carriers and non-carriers of glucocerebrosidase (GBA) mutations. METHODS: EEG was recorded during a visual oddball task in eight Ashkenazi Jewish DLB patients with the N370S mutation in theGBAgene (GBA-DLB) and eleven DLB non-carriers. The time-frequency power and inter-trial phase clustering were calculated from the Morlet wavelet convolution for the midline electrodes. RESULTS: Task performance and cognitive assessments were comparable between groups. While the within-non-GBA-DLB group analysis revealed delta-band power synchronization relative to the baseline (p = 0.01, Cohen's d = 1.0), the within-GBA-DLB-group analysis detected no event-related changes in power. Both groups showed an increase relative to the baseline in the delta and theta bands inter-trial phase clustering (all p < 0.03, d > 1.3). The between-group analysis revealed that event-related power - but not clustering - was lower in GBA-DLB compared to non-carriers in the delta band at Fz and Cz (p = 0.04, d = -0.9). CONCLUSIONS: GBA-DLB patients showed decreased delta-band power compared to non-carriers despite the similar cognitive performance, whereas inter-trial phase clustering was comparable in both groups. SIGNIFICANCE: Preserved inter-trial phase clustering possibly compensates for the impaired power by eliciting the appropriate functional configuration needed for stimulus processing and task performance.

Experience and choice shape expected aversive outcomes.

The value assigned to aversive events is susceptible to contextual influences. Here, we asked whether a change in the valuation of negative events is reflected in an altered neuronal representation of their expected aversive outcome. We show that experiencing an aversive event in the past, and choosing to experience it in the future, reduces its aversive value. This psychological change is mirrored in an altered neural representation of aversive value in the caudate nucleus and anterior cingulate cortex. Our findings indicate that subcortical regions known to track expected value such as the caudate nucleus, together with anterior cingulate cortical regions implicated in emotional modulation, mediate a revaluation in expectancies of aversive states. The results provide a striking example of a contextual sensitivity in how the brain ascribes value to events, in a manner that may foster resilience in the face of adversity.

Dopamine, time, and impulsivity in humans.

Disordered dopamine neurotransmission is implicated in mediating impulsiveness across a range of behaviors and disorders including addiction, compulsive gambling, attention-deficit/hyperactivity disorder, and dopamine dysregulation syndrome. Whereas existing theories of dopamine function highlight mechanisms based on aberrant reward learning or behavioral disinhibition, they do not offer an adequate account of the pathological hypersensitivity to temporal delay that forms a crucial behavioral phenotype seen in these disorders. Here we provide evidence that a role for dopamine in controlling the relationship between the timing of future rewards and their subjective value can bridge this explanatory gap. Using an intertemporal choice task, we demonstrate that pharmacologically enhancing dopamine activity increases impulsivity by enhancing the diminutive influence of increasing delay on reward value (temporal discounting) and its corresponding neural representation in the striatum. This leads to a state of excessive discounting of temporally distant, relative to sooner, rewards. Thus our findings reveal a novel mechanism by which dopamine influences human decision-making that can account for behavioral aberrations associated with a hyperfunctioning dopamine system.

Associations between visual hallucinations and impaired visuo-spatial abilities in dementia with Lewy bodies.

Objectives: In dementia with Lewy bodies (DLB) recurrent visual hallucinations (VH) often coexist or occur consecutively to impaired visual perception. Since in-depth neuropsychological testing is time-consuming, and therefore, not routinely performed in clinical settings, we aimed to explore whether standard cognitive screening tests may be helpful to alert a clinician to the presence of VH in DLB by exploring association between visuo-spatial dysfunction and VH. Method: The clock drawing, cube, and pentagons copying items from Montreal Cognitive Assessment and Mini-Mental State Exam and nonmotor Hooper visual organization test (HVOT) have been scored in DLB patients with and without VH using traditional and extended scoring methods. Results: Forty-five of 69 (65%) DLB patients were VH-positive (VH+). VH+ patients performed worse on the clock drawing (8.8/16 vs. 11.9/16, p = .016) with a higher rate of misrepresentation of time (69% vs. 29%, p = .002) and numbers (53% vs. 25%, p = .024). Likewise, VH+ patients performed worse on the HVOT (13.3/30 vs. 15.7/30, p = .009) having more isolated (6.2/30 vs. 4.4/30, p = .012) types of responses compared to VH- patients. Both groups had similar copying ability (p > .05). The VH discriminative accuracy of the clock drawing was comparable to that of the more elaborate test of visual perception, the HVOT. Conclusions: In DLB impaired drawing and visual organization, but not copying ability is associated with the presence of VH. The simple clock drawing test can be helpful to alert a clinician to the possibility of VH in DLB. (PsycInfo Database Record (c) 2021 APA, all rights reserved).