The cerebellar ataxias (CAs) are a heterogeneous group of disorders characterized by progressive incoordination. Seventeen repeat expansion (RE) loci have been identified as the primary genetic cause and account for >80% of genetic diagnoses. Despite this, diagnostic testing is limited and inefficient, often utilizing single gene assays. This study evaluates the effectiveness of long- and short-read sequencing as diagnostic tools for CA. We recruited 110 individuals (48 females, 62 males) with a clinical diagnosis of CA. Short-read genome sequencing (SR-GS) was performed to identify pathogenic RE and also non-RE variants in 356 genes associated with CA. Independently, long-read sequencing with adaptive sampling (LR-AS) was performed to identify pathogenic RE. SR-GS provided a genetic diagnosis for 38% of the cohort (40/110) including seven non-RE pathogenic variants. RE causes disease in 33 individuals, with the most common condition being SCA27B ( n = 24). In comparison, LR-AS identified pathogenic RE in 29 individuals. RE identification for the two methods was concordant apart from four SCA27B cases not detected by LR-AS due to low read depth. For both technologies manual review of the RE alignment enhances diagnostic outcomes. Orthogonal testing for SCA27B revealed a 15% and 0% false positive rate for SR-GS and LR-AS, respectively. In conclusion, both technologies are powerful screening tools for CA. SR-GS is a mature technology currently used by diagnostic providers, requiring only minor changes in bioinformatic workflows to enable CA diagnostics. LR-AS offers considerable advantages in the context of RE detection and characterization but requires optimization before clinical implementation.

Pharmacokinetic limitations associated with oral ivermectin may limit its success as a potential COVID-19 treatment based on in vitro experiments which demonstrate antiviral efficacy against SARS-CoV-2 at high concentrations. Targeted delivery to the lungs is a practical way to overcome these limitations and ensure the presence of a therapeutic concentration of the drug in a clinically critical site of viral pathology. In this study, the pharmacokinetics (PK) and safety of inhaled dry powders of ivermectin with lactose were investigated in healthy mice. Female BALB/c mice received ivermectin formulation by intratracheal administration at high (3.15 mg/kg) or low doses (2.04 mg/kg). Plasma, bronchoalveolar lavage fluid (BALF), lung, kidney, liver, and spleen were collected at predetermined time points up to 48 h and analyzed for PK. Histological evaluation of lungs was used to examine the safety of the formulation. Inhalation delivery of ivermectin formulation showed improved pharmacokinetic performance as it avoided protein binding encountered in systemic delivery and maintained a high exposure above the in vitro antiviral concentration in the respiratory tract for at least 24 h. The local toxicity was mild with less than 20% of the lung showing histological damage at 24 h, which resolved to 10% by 48 h.

Background: Ivermectin has received worldwide attention as a potential COVID-19 treatment after showing antiviral activity against SARS-CoV-2 in vitro. However, the pharmacokinetic limitations associated with oral administration have been postulated as limiting factors to its bioavailability and efficacy. These limitations can be overcome by targeted delivery to the lungs. In this study, inhalable dry powders of ivermectin and lactose crystals were prepared and characterized for the potential treatment of COVID-19. Methods: Ivermectin was co-spray dried with lactose monohydrate crystals and conditioned by storage at two different relative humidity points (43% and 58% RH) for a week. The in vitro dispersion performance of the stored powders was examined using a medium-high resistance Osmohaler connecting to a next-generation impactor at 60 L/min flow rate. The solid-state characteristics including particle size distribution and morphology, crystallinity, and moisture sorption profiles of raw and spray-dried ivermectin samples were assessed by laser diffraction, scanning electron microscopy, Raman spectroscopy, X-ray powder diffraction, thermogravimetric analysis, differential scanning calorimetry, and dynamic vapor sorption. Results: All the freshly spray-dried formulation (T0) and the conditioned samples could achieve the anticipated therapeutic dose with fine particle dose of 300 μg, FPFrecovered of 70%, and FPFemitted of 83%. In addition, the formulations showed a similar volume median diameter of 4.3 μm and span of 1.9. The spray-dried formulations were stable even after conditioning and exposing to different RH points as ivermectin remained amorphous with predominantly crystalline lactose. Conclusion: An inhalable and stable dry powder of ivermectin and lactose crystals was successfully formulated. This powder inhaler ivermectin candidate therapy appears to be able to deliver doses that could be safe and effective to treat the SARS-COV-2 infection. Further development of this therapy is warranted.

ABSTRACTIntroductionIvermectin is a commonly used antihelminthic agent with over 35 years of established safety data in humans. Recent data demonstrates antiviral activity in vitro against SARS-CoV-2, in addition to a range of viruses. In vitro and animal models also provide evidence of immunomodulatory action. These additional modes of action are supported by in silico modelling, which propose a number of viral and host targets that would mediate these effects.ObjectivesThe aim of this study is to systematically review the published and preprint clinical literature and study results that assessed the potential role of ivermectin as a COVID-19 therapeutic and prophylactic agent.MethodsWe conducted a comprehensive review of PubMed, medRxiv, ClinicalTrials.gov, Global Coronavirus COVID-19 Clinical Trial Tracker, World Health Organization International Clinical Trials Registry Platform, EU Clinical Trials Register, ANZ clinical trials registry, and references from relevant articles.ResultsSearch keywords- “COVID-19 (and synonyms) AND ivermectin”- generated 86 articles on PubMed, 48 on medRvix and 37 on clinicaltrials.gov at the time of writing. Twelve of these were listed as completed clinical trials and of these, 8 were included as investigators had released results. Positive mortality benefit, reduced time to clinical recovery, reduced incidence of disease progression and decreased duration of hospital admission were reported in patients across all stages of clinical severity.LimitationsDue to the time-critical nature of the COVID-19 pandemic our review included preprint data, which must be interpreted with caution while it awaits peer review.

D1-dopamine receptors (Drd1a) are highly expressed in the deep layers of the cerebral cortex and the striatum. A number of human diseases such as Huntington disease and schizophrenia are known to have cortical pathology involving dopamine receptor expressing neurons. To illuminate their functional role, we exploited a Cre/Lox molecular paradigm to generate Emx-1(tox) MUT mice, a transgenic line in which cortical Drd1a-expressing pyramidal neurons were selectively ablated. Emx-1(tox) MUT mice displayed prominent forelimb dystonia, hyperkinesia, ataxia on rotarod testing, heightened anxiety-like behavior, and age-dependent abnormalities in a test of social interaction. The latter occurred in the context of normal working memory on testing in the Y-maze and for novel object recognition. Some motor and behavioral abnormalities in Emx-1(tox) MUT mice overlapped with those in CamKIIα(tox) MUT transgenic mice, a line in which both striatal and cortical Drd1a-expressing cells were ablated. Although Emx-1(tox) MUT mice had normal striatal anatomy, both Emx-1(tox) MUT and CamKIIα(tox) MUT mice displayed selective neuronal loss in cortical layers V and VI. This study shows that loss of cortical Drd1a-expressing cells is sufficient to produce deficits in multiple motor and behavioral domains, independent of striatal mechanisms. Primary cortical changes in the D1 dopamine receptor compartment are therefore likely to model a number of core clinical features in disorders such as Huntington disease and schizophrenia.

Prenatal nicotine exposure alters breathing and ventilatory responses to stress through stimulation of nicotine acetylcholine receptors (nAChRs). We tested the hypothesis that α4-containing nAChRs are involved in mediating the effects of prenatal nicotine exposure on ventilatory and metabolic responses to intermittent mild asphyxia (MA). Using open-flow plethysmography, we measured ventilation (V̇(E)) and rate of O2 consumption ( V̇(O2)) of wild-type (WT) and α4-knock-out (KO) mice, at postnatal (P) days 1-2 and 7-8, with and without prenatal nicotine exposure (6 mg kg(-1) day(-1) beginning on embryonic day 14). Mice were exposed to seven 2 min cycles of mild asphyxia (10% O2 and 5% CO2), each interspersed with 2 min of air. Compared to WT, α4 KO mice had increased air V̇(E) and V̇(O2) at P7-8, but not P1-2. Irrespective of age, genotype had no effect on the hyperventilatory response (increase in V̇(E)/V̇(O2)) to MA. At P1-2, nicotine suppressed air V̇(E) and V̇(O2) in both genotypes but did not affect the hyperventilatory response to MA. At P7-8 nicotine suppressed air V̇(E) and V̇(O2) of only α4 KO's but also significantly enhanced V̇(E) during MA (nearly double that of WT; p<0.001). This study has revealed complex effects of α4 nAChR deficiency and prenatal nicotine exposure on ventilatory and metabolic interactions and responses to stress.

You are free: • to copy, distribute, display, and perform the work. • to make derivative works. Under the following conditions: • Attribution — You must give the original author credit. • Non-Commercial — You may not use this work for commercial purposes. • Share Alike — If you alter, transform, or build upon this work, you may distribute the resulting work only under a licence identical to this one. For any reuse or distribution, you must make clear to others the licence terms of this work. Any of these conditions can be waived if you get permission from the author. Your fair use and other rights are in no way affected by the above. 3 Summary Recent reports have shown that the selective dopamine D 1-like agonist SKF 83822 [which stimulates adenylate cyclase, but not phospholipase C] induces prominent behavioral seizures in mice, whereas its benzazepine congener SKF 83959 [which stimulates phospholipase C, but not adenylate cyclase] does not. To investigate the relative involvement of D 1 vs D 5 receptors in mediating seizures, ethological behavioral topography and cortical EEGs were recorded in D 1 , D 5 and DARPP-32 knockout mice in response to a convulsant dose of SKF 83822. SKF 83822-induced behavioral and EEG seizures were gene dose-dependently abolished in D 1 knockouts. In both heterozygous and homozygous D 5 knockouts, the latency to first seizure was significantly increased and total EEG seizures were reduced relative to wild-types. The majority (60%) of homozygous DARPP-32 knockouts did not have seizures; of those having seizures (40%), the latency to first seizure was significantly increased and the number of high amplitude, high frequency polyspike EEG events was reduced. In addition, immunoblotting was performed to investigate downstream intracellular signalling mechanisms at D 1-like receptors following challenge with SKF 83822 and SKF 83959. In wild-types administered SKF 83822, levels of ERK1/2 and GluR1 AMPA receptor phosphorylation increased twofold in both the striatum and hippocampus; in striatal slices DARPP-32 phosphorylation at Thr34 increased five-fold relative to vehicle-treated controls. These findings indicate that D 1 , and to a lesser extent D 5 , receptor coupling to DARPP-32, ERK1/2 and glutamatergic signalling is involved in mediating the convulsant effects of SKF 83822.

Progressive cell loss is observed in the striatum, cerebral cortex, thalamus, hypothalamus, subthalamic nucleus and hippocampus in Huntington disease. In the striatum, dopamine-responsive medium spiny neurons are preferentially lost. Clinical features include involuntary movements, gait and orofacial impairments in addition to cognitive deficits and psychosis, anxiety and mood disorders. We utilized the Cre–LoxP system to generate mutant mice with selective postnatal ablation of D1 dopamine receptor-expressing striatal neurons to determine which elements of the complex Huntington disease phenotype relate to loss of this neuronal subpopulation.Mutant mice had reduced body weight, locomotor slowing, reduced rearing, ataxia, a short stride length wide-based erratic gait, impairment in orofacial movements and displayed haloperidol-suppressible tic-like movements. The mutation was associated with an anxiolytic profile. Mutant mice had significant striatal-specific atrophy and astrogliosis. D1-expressing cell number was reduced throughout the rostrocaudal extent of the dorsal striatum consistent with partial destruction of the striatonigral pathway. Additional striatal changes included up-regulated D2 and enkephalin mRNA, and an increased density of D2 and preproenkephalin-expressing projection neurons, and striatal neuropeptide Y and cholinergic interneurons. These data suggest that striatal D1-cell-ablation alone may account for the involuntary movements and locomotor, balance and orofacial deficits seen not only in HD but also in HD phenocopy syndromes with striatal atrophy. Therapeutic strategies would therefore need to target striatal D1 cells to ameliorate deficits especially when the clinical presentation is dominated by a bradykinetic/ataxic phenotype with involuntary movements.

Tobacco smoking is a major cause of death and disease and as such there is a critical need for the development of new therapeutic approaches to treat nicotine addiction. Here, we utilize genetic and pharmacological tools to further investigate the nicotinic acetylcholine receptor (nAChR) subtypes that support intravenous self-administration of nicotine. α4-S248F mice contain a point mutation within the α4 nAChR subunit which confers increased sensitivity to nicotine and resistance to mecamylamine. Here, we show that acute administration of mecamylamine (2 mg/kg, i.p.) reduces established nicotine self-administration (0.05 mg/kg/infusion) in wild-type (WT), but not in α4-S248F heterozygous mice, demonstrating a role for α4* nAChRs in the modulation of ongoing nicotine self-administration. Administration of N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), a selective α6β2* nAChR antagonist, dose dependently (5 and 10 mg/kg, i.p.) impairs the acquisition of nicotine self-administration and reduces established nicotine self-administration in WT mice when administered acutely (10 mg/kg, i.p.). This was not due to a general reduction in locomotor activity and the same dose of bPiDI did not affect operant responding for sucrose. bPiDI treatment (10 mg/kg, i.p.) also impaired both the acquisition and maintenance of nicotine self-administration in α4-S248F heterozygous mice. This provides further evidence for the involvement of α6β2* nAChRs in the reinforcing effects of nicotine that underlies its ability to support ongoing self-administration. Taken together, selective targeting of α6β2* or α4α6β2* nAChRs may prove to be an effective strategy for the development of smoking cessation therapies.

Individuals with severe mental illness frequently have difficulties in obtaining and maintaining adequate accommodation. If they are not willing or able to adapt to requirements of traditional supported housing institutions they may live in sheltered and emergency accommodation. Adequate mental health services are rarely available in these facilities. The aim of the present study was to evaluate mental health, functional and social status of individuals living in community sheltered housing facilities. A cross-sectional survey of n=338 individuals in sheltered housing compared to a sample of patients at intake in acute inpatient psychiatry (n=619) concerning clinical and social variables was carried out in the catchment area of Zurich. Matched subsamples of individuals with schizophrenia (n=168) were compared concerning functioning and impairments on the Health of the Nation Outcome Scales (HoNOS). Individuals with schizophrenia in sheltered housing (25% of the residents) have significantly more problems concerning substance use, physical illness, psychopathological symptoms other than psychosis and depression, and relationships, daily activities and occupation than patients with schizophrenia at intake on an acute psychiatric ward. Community sheltered accommodation although conceptualized to prevent homelessness in the general population de facto serve as housing facilities for individuals with schizophrenia and other severe mental illness.

Considerable topographic overlap exists between brain opioidergic and dopaminergic neurons. Pharmacological blockade of the dopamine D1 receptor (Drd1a) reverses several behavioural phenomena elicited by opioids. The present study examines the effects of morphine in adult mutant (MUT) mice expressing the attenuated diphtheria toxin-176 gene in Drd1a-expressing cells, a mutant line shown previously to undergo post-natal striatal atrophy and loss of Drd1a-expression. MUT and wild-type mice were assessed behaviourally following acute administration of 10 mg/kg morphine. Treatment with morphine reduced locomotion and rearing similarly in both genotypes but reduced total grooming only in MUT mice. Morphine-induced Straub tail and stillness were heightened in MUT mice. Chewing and sifting were decreased in MUT mice and these effects were not modified by morphine. Loss of striatal Drd1-positive cells and up-regulated D2-expression, as reflected in down-regulated D1-like and up-regulated D2-like binding, respectively, is not uniform along the cranio-caudal extent in this model but appears to be greater in the caudal striatum. Preferential caudal loss of μ-opioid-expression, a marker for the striosomal compartment, was seen. These data indicate that Drd1a-positive cell loss modifies the exploratory behavioural response elicited by morphine, unmasking novel morphine-induced MUT-specific behaviours and generating a hypersensitivity to morphine for others.

Background Mice generated by a Cre/LoxP transgenic paradigm were used to model neurodegenerative basal ganglia disease of which Huntington disease (HD) is the prototypical example. In HD, death occurs in striatal projection neurons as well as cortical neurons. Cortical and striatal neurons that express the D1 dopamine receptor (Drd1a) degenerate in HD. The contribution that death of specific neuronal cell populations makes to the HD disease phenotype and the response of the brain to loss of defined cell subtypes is largely unknown. Methods Drd1a-expressing cells were targeted for cell death and three independent lines generated; a striatal-restricted line, a cortical-restricted line and a global line in which Drd1a cells were deleted from both the striatum and cortex. Two independent experimental approaches were used. In the first, the proliferative marker Ki-67 was used to identify proliferating cells in eighty-week-old mice belonging to a generic global line, a global in which Drd1a cells express green fluorescent protein (GFP-global) and in eighty-week-old mice of a cortical line. In the second experiment, the proliferative response of four-week-old mice belonging to GFP-global and striatal lines was assessed using the thymidine analogue BrdU. The phenotype of proliferating cells was ascertained by double staining for BrdU and Olig2 (an oligodendrocyte marker), Iba1 (a microglial cell marker), S100β (an astroglial cell marker), or NeuN (a neuronal cell marker). Results In the first study, we found that Ki-67-expressing cells were restricted to the striatal side of the lateral ventricles. Control mice had a greater number of Ki-67+ cells than mutant mice. There was no overlap between Ki-67 and GFP staining in control or mutant mice, suggesting that cells did not undergo cell division once they acquired a Drd1a phenotype. In contrast, in the second study we found that BrdU+ cells were identified throughout the cortex, striatum and periventricular region of control and mutant mice. Mutant mice from the GFP-global line showed increased BrdU+ cells in the cortex, striatum and periventricular region relative to control. Striatal line mutant mice had an increased number of BrdU+ cells in the striatum and periventricular region, but not the cortex. The number of microglia, astrocytes, oligodendrocytes and neurons generated from dividing progenitors was increased relative to control mice in most brain regions in mutant mice from the GFP-global line. In contrast, striatal line mutant mice displayed an increase only in the number of dividing microglia in striatal and periventricular regions. Conclusions Genetically programmed post-natal ablation of Drd1a-expressing neurons is associated with an extensive proliferative response involving multiple cell lineages. The nature of the tissue response has the potential not only to remove cellular debris but also to forge physiologically meaningful brain repair. Age related deficits in proliferation are seen in mutant lines. A blunted endogenous reparative response may underlie the cumulative deficits characteristic of age related neurodegeneration.

Among numerous mechanisms implicated in the regulation of orofacial movements, dopamine-containing neurons have received the most extensive study. Here we review the effects of a) constitutive knockout of D(1-5) dopamine receptors and b) conditional mutations with progressive ablation of D(1) receptor-expressing cells, on the topography of spontaneous and D(1)-like agonist-induced orofacial movements. In constitutive knockouts, D(1) and D(2) exert primary roles in regulating horizontal and vertical jaw movements, respectively, in opposite directions; in contrast, both D(1) and D(2) receptors regulate tongue protrusions and incisor chattering, in the same direction. D(3) and D(5) receptors play more subtle roles in regulating orofacial movements, while D(4) receptors do not play any material role. Progressive loss of forebrain D(1) receptor-expressing cells in CamKIIa/Cre D(1)Tox mutants is associated primarily with decreases in head and vibrissae movements, while progressive loss of striatal D(1) receptor-expressing cells in DARPP-32/Cre D(1)Tox mutants is associated primarily with reductions in jaw movements and tongue protrusions but increases in head and vibrissae movements. Further application of constitutive and particularly conditional mutants may clarify further not only dopaminergic regulation of orofacial movements but also the pathophysiology of orofacial dysfunction in Huntington's disease and Parkinson's disease.

There is a need for objective measures of dyskinesia and bradykinesia of Parkinson's disease (PD) that are continuous throughout the day and related to levodopa dosing. The output of an algorithm that calculates dyskinesia and bradykinesia scores every two minutes over 10 days (PKG: Global Kinetics Corporation) was compared with conventional rating scales for PD in PD subjects. The algorithm recognises bradykinesia as movements made with lower acceleration and amplitude and with longer intervals between movement. Similarly the algorithm recognises dyskinesia as having movements of normal amplitude and acceleration but with shorter periods without movement. The distribution of the bradykinesia and dyskinesia scores from PD subjects differed from that of normal subjects. The algorithm predicted the clinical dyskinesia rating scale AIMS with a 95% margin of error of 3.2 units compared with the inter-rater 95% limits of agreement from 3 neurologists of -3.4 to +4.3 units. Similarly the algorithm predicted the UPDRS III score with a margin of error similar to the inter-rater limits of agreement. Improvement in scores in response to changes in medication could be assessed statistically in individual patients. This algorithm provides objective, continuous and automated assessment of the clinical features of bradykinesia and dyskinesia in PD.

Background: The noninvasive evaluation of nigrostriatal dopaminergic integrity by PET can provide useful information for the differential diagnosis between dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD). Objectives: To evaluate the diagnostic potential of imaging striatal monoaminergic terminal integrity with the novel vesicular monoamine transporter type 2 (VMAT2) radioligand [18F]AV-133 and PET to distinguish DLB from AD. Methods: Fifty participants [9 DLB, 11 AD, 20 Parkinson’s disease (PD) and 10 healthy age-matched control subjects (HC)] underwent [18F]AV-133 PET studies. Additionally, 20 participants underwent amyloid imaging PET scans with either [11C]PiB or 18F-florbetaben. VMAT2 density was calculated through normalized tissue uptake value ratios (RT) at 120–140 min after injection using the primary visual or the cerebellar cortex as reference region. Comparison of the RT for [18F]AV-133 was done between the different clinical diagnostic groups. Results: Significantly lower striatal VMAT2 densities were observed in DLB and PD when compared to AD and HC, especially in the posterior putamen. In contrast to PD and DLB, no reductions were observed in AD patients when compared to HC. Conclusions: [18F]AV-133 allows assessment of nigrostriatal degeneration in Lewy body diseases. In contrast to amyloid imaging, VMAT2 imaging with [18F]AV-133 can robustly detect reductions of dopaminergic nigrostriatal afferents in DLB patients, assisting in the differential diagnosis from AD.

Here we utilize a mouse line with a targeted deletion of the α4 subunit (α4−/− mice), to investigate the role of α4* nAChRs in reinforcing and locomotor effects of nicotine. Within a conditioned place preference paradigm, both α4−/− mice and wild-type (WT) littermates showed a similar place preference to nicotine (0.5 mg/kg i.p.) conditioning. When assessed for operant intravenous self-administration of nicotine (0.05 mg/kg/infusion), α4−/− mice did not differ from their WT littermates in self-administration behavior. To further examine a modulatory role for α4* nAChRs in the reinforcing effects of nicotine, a transgenic mouse with a point mutation of the α4 subunit (α4-S248F) that renders increased sensitivity to low dose nicotine, was assessed for nicotine self-administration over a range of doses. At higher doses examined (0.05 and 0.07 mg/kg/infusion) there was no difference in intravenous nicotine self-administration; however, when mice were offered a lower dose of nicotine (0.03 mg/kg/infusion), α4-S248F mice showed greater nicotine intake than controls. Acute administration of 0.5 mg/kg nicotine caused significant locomotor depression in WT mice but α4−/− mice instead showed significant hyperactivity. Following chronic, intermittent administration of this dose of nicotine only WT mice displayed significant tolerance. Analogous experiments utilizing administration of the nicotinic antagonist mecamylamine in WT mice confirmed a dissociation between the putative nicotinic receptor subtypes required for mediating psychomotor and reinforcing effects of nicotine. These data demonstrate a necessary role for α4* nAChRs in the locomotor depressant effect of nicotine but not the reinforcing effects that support ongoing self-administration of nicotine.