α-Synuclein Pathology in PRKN-Linked Parkinson's Disease: New Insights from a Blood-Based Seed Amplification Assay.

Pathogenic variants in PRKN cause early-onset Parkinson's disease (PD), while the role of alpha-synuclein in PRKN-PD remains uncertain. One study performed a blood-based alpha-synuclein seed amplification assay (SAA) in PRKN-PD, not detecting seed amplification in 17 PRKN-PD patients. By applying a methodologically different SAA focusing on neuron-derived extracellular vesicles, we demonstrated alpha-synuclein seed amplification in 8 of 13 PRKN-PD patients, challenging the view of PRKN-PD as a non-synucleinopathy. Moreover, we performed blinded replication of the neuron-derived extracellular vesicles-dependent SAA in idiopathic PD patients and healthy controls. In conclusion, blood-based neuron-derived extracellular vesicles-dependent SAA represents a promising biomarker to elucidate the underpinnings of (monogenic) PD. ANN NEUROL 2024;95:1173-1177.

LRRK2 activation controls the repair of damaged endomembranes in macrophages.

Cells respond to endolysosome damage by either repairing the damage or targeting damaged endolysosomes for degradation via lysophagy. However, the signals regulating the decision for repair or lysophagy are poorly characterised. Here, we show that the Parkinson's disease (PD)-related kinase LRRK2 is activated in macrophages by pathogen- or sterile-induced endomembrane damage. LRRK2 recruits the Rab GTPase Rab8A to damaged endolysosomes as well as the ESCRT-III component CHMP4B, thereby favouring ESCRT-mediated repair. Conversely, in the absence of LRRK2 and Rab8A, damaged endolysosomes are targeted to lysophagy. These observations are recapitulated in macrophages from PD patients where pathogenic LRRK2 gain-of-function mutations result in the accumulation of endolysosomes which are positive for the membrane damage marker Galectin-3. Altogether, this work indicates that LRRK2 regulates endolysosomal homeostasis by controlling the balance between membrane repair and organelle replacement, uncovering an unexpected function for LRRK2, and providing a new link between membrane damage and PD.

Mitochondrial proteins encoded by the 22q11.2 neurodevelopmental locus regulate neural stem and progenitor cell proliferation.

Microdeletion of a 3Mb region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes individuals to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha, encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Single mutant analysis shows that both mrpl40 and prodha mutants display aberrant neural stem and progenitor cell proliferation, with each gene regulating distinct cell populations. Finally, double mutants for both mrpl40 and prodha display aggravated behavioral phenotypes and neural stem and progenitor cell analysis reveals a previously unrecognized partially redundant role for mrpl40 and prodha in regulating radial glia-like cell proliferation. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.

A comparison between erythrocytapheresis and venesection for the treatment of JAK2-mutated polycythaemia.

BACKGROUND: JAK2-mutated polycythaemia vera (PV) is associated with reduced survival because of thrombotic events and haematological disease transformation. Therapeutic venesection has traditionally been used to lower haematocrit, but the technique of erythrocytapheresis has emerged over the last decade. AIM: To compare erythrocytapheresis with venesection as treatment for PV by assessing medical efficacy and financial viability. METHODS: One hundred sixteen patients with PV who received red cell depletion therapy at Barwon Health between 2014 and 2021 were identified. The haematocrit drop after each session, interval between treatment times and number of sessions required to achieve a haematocrit <0.45 were compared with an independent t test. Thrombosis rates were compared with Pearson's chi-squared test. Cost-funding analysis was done by assessing the Weighted Inlier Equivalent Separation and National Weighted Activity Unit funding models. RESULTS: Patients treated with erythrocytapheresis achieved a greater haematocrit drop each treatment session (0.075 vs 0.03, P < 0.01), required fewer sessions to achieve a haematocrit <0.45 (1 vs 4, P < 0.01) and experienced fewer thrombotic complications (8.7% vs 32.1%, P = 0.02) than those treated with venesection. Cost-funding analysis demonstrated that erythrocytapheresis was more financially viable with a surplus of AU$297 per session compared to a deficit of AU$176 with venesection. Even if funding for venesection is increased, the cost of erythrocytapheresis may be mitigated by a lower number of procedures required per year (3.8 vs 5.3, P < 0.01). CONCLUSIONS: Erythrocytapheresis is more efficacious than venesection for the treatment of PV and is accompanied by rapid reductions in haematocrit and reduced thrombotic complications.

Indium-labelled autologous platelet sequestration studies predict response to splenectomy in immune thrombocytopenia: an Australian experience.

BACKGROUND: Splenectomy is an effective intervention in primary immune thrombocytopenia (ITP). Attempts to define pre-clinical predictors of platelet response to splenectomy are inconsistent. Based on international studies defining the likelihood of platelet response using platelet sequestration, patients with relapsed/refractory ITP being considered for splenectomy at a regional Australian hospital were assessed with 111 indium-labelled autologous platelet sequestration (ILAPS) studies. AIMS: To audit the use of ILAPS in an Australian setting and define its role in predicting response to splenectomy. METHODS: A retrospective review of all patients referred for an ILAPS study at a regional hospital was performed. Results for each patient were expressed as an 'R' value (spleen/ liver uptake ratio) to quantify the platelet sequestration pattern and outcome post-splenectomy, based on platelet counts. RESULTS: A total of 45 patients was identified: 13 underwent splenectomy and 32 were medically managed. Patients with favourable ILAPS scans (pure or predominant splenic sequestration) demonstrated a superior response post-splenectomy (100% overall response rate (ORR); 83.5% complete remission (CR)) compared with those with unfavourable ILAPS scans (mixed or pure hepatic sequestration) (71.4% ORR; 57.1% CR) over 12 months. CONCLUSIONS: The use of ILAPS in the Australian setting is feasible and this experience confirms larger international studies demonstrating its utility as a predictor of response to splenectomy in ITP. An unfavourable ILAPS scan could be considered a negative predictor of response prompting consideration for other emerging ITP treatments such as thrombopoietin-receptor agonists or B-cell depleting therapy such as Rituximab.

Overall survival with daratumumab, bortezomib, melphalan, and prednisone in newly diagnosed multiple myeloma (ALCYONE): a randomised, open-label, phase 3 trial.

BACKGROUND: Standard-of-care treatment for patients with newly diagnosed multiple myeloma includes combination therapies for patients who are not eligible for autologous stem-cell transplantation. At the primary analysis for progression-free survival of the phase 3 ALCYONE trial, progression-free survival was significantly longer with daratumumab in combination with bortezomib, melphalan, and prednisone (D-VMP) versus bortezomib, melphalan, and prednisone (VMP) alone in patients with transplant-ineligible, newly diagnosed multiple myeloma. Here we report updated efficacy and safety results from a prespecified, interim, overall survival analysis of ALCYONE with more than 36 months of follow-up. METHODS: ALCYONE was a multicentre, randomised, open-label, active-controlled, phase 3 trial that enrolled patients between Feb 9, 2015, and July 14, 2016, at 162 sites in 25 countries across North America, South America, Europe, and the Asia-Pacific region. Patients were eligible for inclusion if they had newly diagnosed multiple myeloma and were ineligible for high-dose chemotherapy with autologous stem-cell transplantation, because of their age (≥65 years) or because of substantial comorbidities. Patients were randomly assigned in a 1:1 ratio and by permuted block randomisation to receive D-VMP or VMP. An interactive web-based randomisation system was used. Randomisation was stratified by International Staging System disease stage, geographical region, and age. There was no masking to treatment assignments. All patients received up to nine 6-week cycles of subcutaneous bortezomib (1·3 mg/m2 of body surface area on days 1, 4, 8, 11, 22, 25, 29, and 32 of cycle one and on days 1, 8, 22, and 29 of cycles two through nine), oral melphalan (9 mg/m2 once daily on days 1 through 4 of each cycle), and oral prednisone (60 mg/m2 once daily on days 1 through 4 of each cycle). Patients in the D-VMP group also received intravenous daratumumab (16 mg/kg of bodyweight, once weekly during cycle one, once every 3 weeks in cycles two through nine, and once every 4 weeks thereafter as maintenance therapy until disease progression or unacceptable toxicity). The primary endpoint was progression-free survival, which has been reported previously. Results presented are from a prespecified interim analysis for overall survival. The primary analysis population (including for overall survival) was the intention-to-treat population of all patients who were randomly assigned to treatment. The safety population included patients who received any dose of study treatment. This trial is registered with ClinicalTrials.gov, NCT02195479. FINDINGS: 706 patients were randomly assigned to treatment groups (350 to the D-VMP group, 356 to the VMP group). At a median follow-up of 40·1 months (IQR 37·4-43·1), a significant benefit in overall survival was observed for the D-VMP group. The hazard ratio (HR) for death in the D-VMP group compared with the VMP group was 0·60 (95% CI 0·46-0·80; p=0·0003). The Kaplan-Meier estimate of the 36-month rate of overall survival was 78·0% (95% CI 73·2-82·0) in the D-VMP group and 67·9% (62·6-72·6) in the VMP group. Progression-free survival, the primary endpoint, remained significantly improved for the D-VMP group (HR 0·42 [0·34-0·51]; p<0·0001). The most frequent adverse events during maintenance daratumumab monotherapy in patients in the D-VMP group were respiratory infections (54 [19%] of 278 patients had upper respiratory tract infections; 42 [15%] had bronchitis, 34 [12%] had viral upper respiratory tract infections), cough (34 [12%]), and diarrhoea (28 [10%]). INTERPRETATION: D-VMP prolonged overall survival in patients with newly diagnosed multiple myeloma who were ineligible for stem-cell transplantation. With more than 3 years of follow-up, the D-VMP group continued to show significant improvement in progression-free survival, with no new safety concerns. FUNDING: Janssen Research & Development.

Daratumumab plus Bortezomib, Melphalan, and Prednisone for Untreated Myeloma.

BACKGROUND: The combination of bortezomib, melphalan, and prednisone is a standard treatment for patients with newly diagnosed multiple myeloma who are ineligible for autologous stem-cell transplantation. Daratumumab has shown efficacy in combination with standard-of-care regimens in patients with relapsed or refractory multiple myeloma. METHODS: In this phase 3 trial, we randomly assigned 706 patients with newly diagnosed multiple myeloma who were ineligible for stem-cell transplantation to receive nine cycles of bortezomib, melphalan, and prednisone either alone (control group) or with daratumumab (daratumumab group) until disease progression. The primary end point was progression-free survival. RESULTS: At a median follow-up of 16.5 months in a prespecified interim analysis, the 18-month progression-free survival rate was 71.6% (95% confidence interval [CI], 65.5 to 76.8) in the daratumumab group and 50.2% (95% CI, 43.2 to 56.7) in the control group (hazard ratio for disease progression or death, 0.50; 95% CI, 0.38 to 0.65; P<0.001). The overall response rate was 90.9% in the daratumumab group, as compared with 73.9% in the control group (P<0.001), and the rate of complete response or better (including stringent complete response) was 42.6%, versus 24.4% (P<0.001). In the daratumumab group, 22.3% of the patients were negative for minimal residual disease (at a threshold of 1 tumor cell per 105 white cells), as compared with 6.2% of those in the control group (P<0.001). The most common adverse events of grade 3 or 4 were hematologic: neutropenia (in 39.9% of the patients in the daratumumab group and in 38.7% of those in the control group), thrombocytopenia (in 34.4% and 37.6%, respectively), and anemia (in 15.9% and 19.8%, respectively). The rate of grade 3 or 4 infections was 23.1% in the daratumumab group and 14.7% in the control group; the rate of treatment discontinuation due to infections was 0.9% and 1.4%, respectively. Daratumumab-associated infusion-related reactions occurred in 27.7% of the patients. CONCLUSIONS: Among patients with newly diagnosed multiple myeloma who were ineligible for stem-cell transplantation, daratumumab combined with bortezomib, melphalan, and prednisone resulted in a lower risk of disease progression or death than the same regimen without daratumumab. The daratumumab-containing regimen was associated with more grade 3 or 4 infections. (Funded by Janssen Research and Development; ALCYONE ClinicalTrials.gov number, NCT02195479 .).

Health-related quality of life in patients with newly diagnosed multiple myeloma ineligible for stem cell transplantation: results from the randomized phase III ALCYONE trial.

BACKGROUND: In the phase III ALCYONE trial, daratumumab plus bortezomib/melphalan/prednisone (D-VMP) significantly improved overall response rate and progression-free status compared with VMP alone in transplant-ineligible patients with newly diagnosed multiple myeloma (NDMM). Here, we present patient-reported outcomes (PROs) from ALCYONE. METHODS: The European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30-item (EORTC QLQ-C30) and EuroQol 5-dimensional descriptive system (EQ-5D-5L) questionnaire were administered at baseline, every 3 months (year 1) and every 6 months (until progression). Treatment effects were assessed using a repeated-measures, mixed-effects model. RESULTS: Compliance with PRO assessments was comparable at baseline (> 90%) and throughout study (> 76%) for both treatment groups. Improvements from baseline were observed in both groups for EORTC QLQ-C30 Global Health Status (GHS), most functional scales, symptom scales and EQ-5D-5L visual analog scale (VAS). Between-group differences were significant for GHS (p = 0.0240) and VAS (p = 0.0160) at month 3. Improvements in pain were clinically meaningful in both groups at all assessment time points. Cognitive function declined in both groups, but the magnitude of the decline was not clinically meaningful. CONCLUSIONS: Patients with transplant-ineligible NDMM demonstrated early and continuous improvements in health-related quality of life, including improvements in functioning and symptoms, following treatment with D-VMP or VMP. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02195479 , registered September 21, 2014.

Autologous stem cell transplantation in elderly multiple myeloma patients aged ≥65 years: a two-centre Australian experience.

There are currently limited Australian data on the outcomes of autologous stem cell transplantation (ASCT) in elderly multiple myeloma (MM) patients. We present the largest cohort of elderly MM patients aged ≥65 years undergoing ASCT in Australia and report their outcomes based on our two-centre experience. Our study affirms that ASCT is well tolerated, safe and effective in elderly MM patients aged ≥65 years and should be considered an important component of treatment in patients who are fit enough for the procedure.

Correction: Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1006918.].

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis.

During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.

Computer Tomography (CT) for head injury: adherence to the National Institute for Health and Care Excellence (NICE) criteria.

Objective Head injury is a common presentation to the emergency department yet adherence to guidelines for management is suboptimal. Our study assesses adherence to National Institute for Health and Care Excellence (NICE) computed tomography (CT) head guidelines at a teaching hospital in the United Kingdom. It critically evaluates the efficacy of interventions. Methods We performed a quality improvement project to improve adherence to NICE CT head scan guidelines following head injury. Data was collected over one-month in 2014, and 2017. Interventions included education of the multidisciplinary team, information sheets, team engagement and tri-annual education sessions for junior doctors. The success of these interventions in the adult population was assessed objectively during the second cycle and subjectively via a junior doctor survey. Results 171 patients were included in the study. Following interventions, there was a statistically significant decrease of 23% in the number of CT heads requested with no clear indication (P = 0.00027). The mean time to scan for the one-hour indications decreased from 73 to 55 minutes. The mean time to report all scans decreased from 89 to 57 minutes. The survey results were encouraging. Conclusion Adherence to NICE guidelines for head injury is inadequate but can be improved by interventions.

Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis.

In animals, specification of the primordial germ cells (PGCs), the stem cells of the germ line, is required to transmit genetic information from one generation to the next. Bucky ball (Buc) is essential for germ plasm (GP) assembly in oocytes, and its overexpression results in excess PGCs in zebrafish embryos. However, the mechanistic basis for the excess PGCs in response to Buc overexpression, and whether endogenous Buc functions during embryogenesis, are unknown. Here, we show that endogenous Buc, like GP and overexpressed Buc-GFP, accumulates at embryonic cleavage furrows. Furthermore, we show that the maternally expressed zebrafish Kinesin-1 Kif5Ba is a binding partner of Buc and that maternal kif5Ba (Mkif5Ba) plays an essential role in germline specification in vivo. Specifically, Mkif5Ba is required to recruit GP to cleavage furrows and thereby specifies PGCs. Moreover, Mkif5Ba is required to enrich Buc at cleavage furrows and for the ability of Buc to promote excess PGCs, providing mechanistic insight into how Buc functions to assemble embryonic GP. In addition, we show that Mkif5Ba is also essential for dorsoventral (DV) patterning. Specifically, Mkif5Ba promotes formation of the parallel vegetal microtubule array required to asymmetrically position dorsal determinants (DDs) towards the prospective dorsal side. Interestingly, whereas Syntabulin and wnt8a translocation depend on kif5Ba, grip2a translocation does not, providing evidence for two distinct mechanisms by which DDs might be asymmetrically distributed. These studies identify essential roles for maternal Kif5Ba in PGC specification and DV patterning, and provide mechanistic insight into Buc functions during early embryogenesis.

Dynamic visualization of transcription and RNA subcellular localization in zebrafish.

Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the ßactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.