Biology subjects

Milnerwood, A.

Publications and source records attributed to Milnerwood, A..

8 recordsLinked to original sources

bioRxiv2026

Sex- and age-dependent control of dopamine function by LRRK2, and rapid reversal of dopamine release deficits in the LRRK2-G2019S knock-in mouse model of Parkinson's Disease.

No disease-modifying therapy exists for Parkinson's disease (PD). At diagnosis (~65y), ~70% of nigrostriatal dopamine (DA) axons may have degenerated, so identifying when and how striatal axon function declines is critical. LRRK2 hyperactivity is observed in LRRK2 and VPS35 familial cases, and also idiopathic PD. Thus suggests LRRK2 hyperfunction is a common pathophysiological feature of PD. LRRK2 inhibitors are currently in human trials, yet their effects on brain circuit function are poorly characterized. By optical imaging of iGluSnFR and dLight we measured striatal glutamate and DA release in WT, LRRK2 G2019S (LKI) and VPS35 D620N (VKI) knock-in mice, across adult ages and sexes, with and without LRRK2 inhibition. In WT mice, ongoing LRRK2 activity regulated DA release in an age- and sex-dependent manner. Acute inhibition reversed basal sex differences in striatal glutamate and DA release in aged mice, and potentiated DA release more in middle-aged females than males. In LKI mice, DA release declined progressively, first in males, then females, after maturation to full adulthood, and remained impaired in very old LKI and VKI mice. Release deficits in aged LKI and VKI mice were resistant to acute LRRK2 inhibition, but 2h inhibition rapidly reversed deficits in middle-aged LKI males and females. We conclude that LRRK2 activity, and inhibition effects, depend on age, sex, and genotype, with direct implications for the design and timing of current LRRK2 inhibitor clinical trials for PD.

neuroscience↗

bioRxiv2026

Behavioural hyperactivity and risk-taking in young-adult male mice correlate with increased dopamine release in dorsal, but not ventral, striatum

Adolescence is characterized by increased novelty seeking, impulsivity, locomotion and risk-taking, behaviours that facilitate the transition to adulthood but may also increase vulnerability to psychiatric illness. Maturation of dopamine (DA) transmission within the striatum is thought to contribute to these behavioural changes. However, the developmental trajectory of striatal DA release remains unclear, with conflicting evidence regarding age- and sex-dependent changes, particularly across dorsal and ventral striatal regions. Complicating interpretation, many studies classify mice younger than 2 months as 'adults,' potentially obscuring important developmental differences. Here, we examined how age, sex and striatal subregion influence DA release during adolescence and early adulthood. DA release was measured in dorsal and ventral striatal brain slices from male and female C57BL/6J mice at 2 and 4 months of age, corresponding to adolescence and young adulthood. Ultrafast imaging of the genetically encoded DA sensor, dLight, was used to quantify extracellular DA transients evoked by low- and high-frequency electrical stimulation. Glutamatergic and cholinergic transmission was pharmacologically blocked to isolate DA release intrinsic to dopaminergic axons. DA release measures were related to exploratory and locomotor behaviour. We identified a male-specific increase in dorsal striatal DA release between 2 and 4 months of age that coincided with increased locomotion and risk-taking. In contrast, DA release in the ventral striatum did not show comparable age- or sex-dependent changes. These differences were not attributable to altered glutamatergic or cholinergic contributions to DA release. Together, these findings demonstrate that maturation of striatal DA transmission is region- and sex-dependent, with prominent developmental changes occurring in the dorsal striatum during the transition from adolescence to adulthood. These findings help reconcile conflicting reports of developmental DA release and emphasize the importance of considering age, sex and striatal subregion when defining mature dopaminergic function in rodents.

neuroscience↗

bioRxiv2026

Neuronal secretome from bipolar patient-derived neurons alters network function and contains candidate biomarkers for diagnosis and lithium response

Delayed diagnosis and treatment are a major burden to patients with bipolar disorder. While lithium is the most effective treatment against mania, depressive episodes, and suicide, only 30% of patients respond to it fully. Currently there are no reliable methods to predict lithium responsiveness. To address these challenges, we aimed to identify potential diagnostic and treatment response biomarkers for BD, in addition furthering understanding of BD pathophysiology. Here, we leveraged human induced pluripotent stem cell (hiPSC) derived neurons from lithium responsive (LR), lithium non-responsive (LNR), and healthy age-matched controls (CTL). We found extracellular vesicle (EV) cargos from hiPSC-derived neurons are indicative of disease state and treatment-response. Unbiased proteomic and miRNA profiling identified 10 proteins and 13 miRNAs that were differentially expressed in BD EVs relative to CTL, as well as distinct molecular signatures separating LR ad LNR groups. These differences converged on pathways related to synaptic function, neurotrophic signalling, and cellular stress responses. Additionally, we found the BD neuronal secretome alters activity in non-BD neuronal networks. Chronic treatment of CTL cultures with BD neuron-conditioned media modified the proportion of active neurons and the frequency and amplitude of calcium transients in individual neurons. We demonstrate that neuronal EVs contain molecular signatures of disease state and treatment response in BD and identify the BD secretome as an active regulator of neuronal network homeostasis. This study provides novel insights into the pathophysiology of BD and candidate biomarkers for personalized BD diagnosis and treatment selection.

neuroscience↗

bioRxiv2024

LRRK2 G2019S mutation incites increased cell-intrinsic neutrophil effector functions and intestinal inflammation in a model of infectious colitis

Parkinsons Disease (PD) is a progressive, neurodegenerative disorder characterised by motor and non-motor symptoms. Emerging evidence suggests a link between PD and gastrointestinal dysfunction. Constipation is frequently observed years prior to development of motor dysfunction in PD, and people with inflammatory bowel disease (IBD) are more likely to develop PD. Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) account for approximately 1% of all PD cases and are associated with increased risk for IBD. Among them, LRRK2 Gly2019Ser (G2019S), located within the kinase domain, is the most common PD-associated mutation and increases kinase activity. It is unknown how LRRK2 mutation affects susceptibility to intestinal inflammation or pathogenesis of PD. Using single cell RNA sequencing (scRNAseq), we demonstrate that LRRK2 G2019S mutation promotes a dysregulated gene profile, especially within neutrophil, monocyte and {gamma}{delta} T cell populations, following Citrobacter rodentium infection in mice. Transcriptionally, LRRK2 G2019S neutrophils have a greater pro- inflammatory type I and II IFN response compared to those of WT mice. This is accompanied by an increase in neutrophil numbers in the lamina propria in LRRK2 G2019S mice. We also uncover cell-intrinsic functional defects in LRRK2 G2019S neutrophils, including increased chemotaxis, degranulation and neutrophil extracellular traps (NETosis) formation. Increased neutrophil infiltration is associated with an upregulation in Th17 immune responses, which may together contribute to the observed increase in colon pathology during infection. These findings increase our understanding of the role of PD-associated genes in immune cells and their contribution to immune dysregulation. Understanding the early perturbations driven by the LRRK2 G2019S mutation in gastrointestinal pathology may facilitate the development of biomarkers for early diagnosis and intervention in PD.

immunology↗

bioRxiv2024

Emergent glutamate & dopamine dysfunction in VPS35(D620N) knock-in mice and rapid reversal by LRRK2 inhibition

The D620N variant in Vacuolar Protein Sorting 35 (VPS35) causes autosomal-dominant, late- onset Parkinsons disease. VPS35 is a core subunit of the retromer complex that canonically recycles transmembrane cargo from sorting endosomes. Although retromer cargoes include many synaptic proteins, VPS35s neuronal functions are poorly understood. To investigate the consequences of the Parkinsons mutation, striatal neurotransmission was assessed in 1-, 3- & 6-month-old VPS35 D620N knock-in (VKI) mice. Spontaneous and optogenetically- evoked corticostriatal glutamate transmission was increased in VKI striatal spiny projection neurons by 6 months, when total striatal glutamate release, quantified by iGluSnFR imaging, showed similarities to wild-type. dLight imaging revealed robust increases in VKI striatal dopamine release by 6 months, which were reversed with acute ex vivo leucine-rich repeat kinase 2 (LRRK2) inhibition. We conclude that increased glutamate and dopamine transmission in VKI mice progressively emerges in young-adulthood, and that dopamine dysfunction is likely the result of sustained, rapidly-reversible, LRRK2 hyperactivity.

neuroscience↗

bioRxiv2023

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinsons disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

neuroscience↗

bioRxiv2023

Molecular signatures of hyperexcitability and lithium responsiveness in bipolar disorder patient neurons provide alternative therapeutic strategies.

Bipolar disorder (BD) is a multifactorial psychiatric illness affecting about 1% of the world population. The first line treatment, lithium (Li), is effective in only a subset of patients and its mechanism of action remains largely elusive. In the present study, we used iPSC-derived neurons from BD patients responsive (LR) or not (LNR) to lithium and combined electrophysiology, calcium imaging, biochemistry, transcriptomics, and phosphoproteomics to report mechanistic insights into neuronal hyperactivity in BD, and Lis mode of action. We show a selective rescue of neuronal hyperactivity by Li in BD LR neurons through changes in Na+ currents. The whole transcriptome sequencing revealed altered gene expression in BD neurons in pathways related to glutamatergic transmission, and Li selectively altered those involved in cell signaling and ion transport/channel activity. We found the therapeutic effect of Li in BD LR patients was associated with Akt signaling and confirmed that an Akt activator mimics Li effect in BD LR neurons. Further, we showed that AMP-activated protein kinase (AMPK) reduces neural network activity and sodium currents in BD LNR patients. These findings suggest the potential for novel treatment strategies in BD, such as Akt activators in BD LR cases, and the use of AMPK activators for BD LNR patients.

neuroscience↗

bioRxiv2023

Inhibition of LRRK2 kinase activity rescues deficits in striatal dopamine dynamics in VPS35 p.D620N knock-in mice

Dysregulation of dopamine neurotransmission profoundly affects motor, motivation and learning behaviors, and is often observed during the prodromal phase of Parkinsons disease (PD). However, the mechanism underlying these pathophysiological changes remains to be elucidated. Mutations in vacuolar protein sorting 35 (VPS35) and leucine-rich repeat kinase 2 (LRRK2) both lead to autosomal dominant PD, and VPS35 and LRRK2 may physically interact to govern the trafficking of synaptic cargos within the endo-lysosomal network in a kinase-dependent manner. To better understand the functional role of VPS35 and LRRK2 on dopamine physiology, we examined Vps35 haploinsufficient (Haplo) and Vps35 p.D620N knock-in (VKI) mice and how their behavior, dopamine kinetics and biochemistry are influenced by LRRK2 kinase inhibitors. We found Vps35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12 and Rab29. In contrast, Vps35 haploinsufficiency reduces phosphorylation of Rab12. While striatal dopamine transporter (DAT) expression and function is similarly impaired in both VKI and Haplo mice, that physiology is normalized in VKI by treatment with the LRRK2 kinase inhibitor, MLi-2. As a corollary, VKI animals show a significant increase in amphetamine induced hyperlocomotion, compared to Haplo mice, that is also abolished by MLi-2. Taken together, these data show Vps35 p.D620N confers a gain-of-function with respect to LRRK2 kinase activation, and VPS35 and LRRK2 functionally interact to regulate DAT trafficking and striatal dopamine neurotransmission.

neuroscience↗