Objective Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease‐modifying treatments are approved. A key therapeutic target in PD is α‐synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl–coenzyme A desaturase (SCD) inhibitor (“5b”) that prevented αS‐positive vesicular inclusions and cytotoxicity in cultured human neurons. Methods Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild‐type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD‐like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD‐related lipid changes were quantified in 5b‐ versus placebo‐treated mice. Outcomes were compared to effects of crossing 3K to SCD1−/− mice. Results 5b treatment reduced αS hyperphosphorylation in E46K‐expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD‐like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K‐resistant lipid‐rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation. Interpretation Prolonged reduction of brain SCD activity prevented PD‐like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α‐synucleinopathies. ANN NEUROL 2021;89:74–90

Many studies report a higher risk for Parkinson's disease (PD) and younger age of onset in men. This, and the fact that the neuropathological process underlying PD symptoms may begin before menopause, suggests that estrogen-based hormone therapy could modify this higher risk in males. However, the effects of female sex or estrogen on ␣-synuclein (␣S) homeostasis and related PD neuropathology remain unknown. Here, we used an ␣S tetramer-abrogating mouse model of PD (3K) that amplifies the familial E46K PD mutation to investigate the effects of female sex and brain-selective estrogen treatment on ␣S tetramerization and solubility, formation of vesicle-rich ␣S ϩ aggregates, dopaminergic and cortical fiber integrity, and associated motor deficits. In male 3K mice, the motor phenotype became apparent at ϳ10 weeks and increased to age 6 months, paralleled by PD-like neuropathology, whereas 3K females showed a significant delay in onset. At 6 months, this beneficial phenotypic effect in 3K females was associated with a higher ␣S tetramer-to-monomer ratio and less decrease in dopaminergic and cortical fiber length and quantity. Brain-selective estrogen treatment in symptomatic 3K mice significantly increased the tetramer-to-monomer ratio, turnover by autophagy of aggregate-prone monomers, and neurite complexity of surviving DAergic and cortical neurons, in parallel with benefits in motor performance. Our findings support an upstream role for ␣S tetramer loss in PD phenotypes and a role for estrogen in mitigating PD-like neuropathology in vivo. Brain-selective estrogen therapy may be useful in delaying or reducing PD symptoms in men and postmenopausal women.

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson’s disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient–derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer–monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD–causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)–human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

The dendritic arbor of neurons constrains the pool of available synaptic partners and influences the electrical integration of synaptic currents. Despite these critical functions, our knowledge of the dendritic structure of cortical neurons during early postnatal development and how these dendritic structures are modified by visual experience is incomplete. Here, we present a large-scale dataset of 849 3D reconstructions of the basal arbor of pyramidal neurons collected across early postnatal development in visual cortex of mice of either sex. We found that the basal arbor grew substantially between postnatal day 7 (P7) and P30, undergoing a 45% increase in total length. However, the gross number of primary neurites and dendritic segments was largely determined by P7. Growth from P7 to P30 occurred primarily through extension of dendritic segments. Surprisingly, comparisons of dark-reared and typically reared mice revealed that a net gain of only 15% arbor length could be attributed to visual experience; most growth was independent of experience. To examine molecular contributions, we characterized the role of the activity-regulated small GTPase Rem2 in both arbor development and the maintenance of established basal arbors. We showed that Rem2 is an experience-dependent negative regulator of dendritic segment number during the visual critical period. Acute deletion of Rem2 reduced directionality of dendritic arbors. The data presented here establish a highly detailed, quantitative analysis of basal arbor development that we believe has high utility both in understanding circuit development as well as providing a framework for computationalists wishing to generate anatomically accurate neuronal models.

17 65 processes. For the first time, we also establish Rem2 -a small GTPase previously implicated in 66 dendritic complexity in vitro and in Xenopus Laevis optic tectum [51, 53-55] -as an experience-67 dependent negative regulator of dendritic complexity in mammalian visual cortex. This work 68 demonstrates that Rem2 cell-autonomously regulates the arrangement of the basal dendrites: 69 neurons in which Rem2 has been deleted are less likely to exhibit a significant directionality and 70 sometimes exhibit abnormal directionality. Taken together, our results establish a unified 71 framework for investigating dendritic development in the mammalian cortex and expand our 72 understanding of how dendritic arbors are established and maintained in an experience-rich 73 environment. 74 75 Methods 76 Golgi-Cox Labeling and Tissue Preparation 77 Typically-reared WT and Rem2 -/littermate mice were housed in a 12hr light/12hr dark 78 cycle from birth until the specified age (P7, P12, P16, P21, P30). Dark-reared mice were placed 79 in a light-tight box beginning at P9 until termination of the experiment (P16, P21, or P30). At 80 the specified age, mice were anesthetized with ketamine/xylazine cocktail (ketamine 81 100mg/kg, xylazine 10mg/kg) and transcardially perfused with 0.9% saline in ddH20. Dark-82 reared mice were anesthetized in the dark and then shielded from light until after perfusion 83 using a mask constructed of several layers of light blocking tape (Thor Labs) to cover the eyes. 84 Immediately following perfusion, brains were weighed and submerged in Golgi-Cox solution (FD 85 NeuroTechnologies). Throughout all steps involving Golgi-Cox, brains were protected from light. 86 Golgi-Cox solution was changed 24 hours after initial immersion and brains continued to be 87 stored in Golgi-Cox solution for 7 days. After 7 days, brains were transferred to Solution C (FD 88 NeuroTechnologies) for at least 2 days. Coronal sections were cut at 150 µm using a cryostat 89 and immediately mounted on slides coated with 2% porcine gelatin. Histology was carried out 90 according to the protocol supplied by FD NeuroTechnologies RapidGolgi Stain Kit. Briefly, slides 91 were washed with ddH2O, developed using FD Neurotechnologies Solutions D & E, rinsed in 92 ddH2O, dehydrated with a graded series of ethanols, and cleared using xylenes. Slides were 93 then coverslipped using Permount (Fisher Scientific).94 95 TdTomato Reporter Tissue Preparation 96 Typically reared Rem2 +/+ ;TdT flex/flex and Rem2 flx/flx ;TdT flex/flex mice were housed in 12hr 97 light/ 12hr dark cycle from birth until the specified ages and days post injection (dpi) (5dpi/P25, 98 7dpi/P27, 10dpi/P30). Mice (both Rem2 +/+ ; TdT flex/flex and Rem2 flx/flx ; TdT flex/flex ) were injected 99 with AAV.GFP-Cre at P20 (see detailed method below) to activate reporter expression and, in 100 Rem2 flx/flx ; TdT flex/flex mice, manipulate Rem2 expression. At the specified age, mice were 101 anesthetized with ketamine/xylazine cocktail (ketamine 100mg/kg, xylazine 10mg/kg) and 1...

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