LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory.

Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction.

Memory and synaptic deficits in Hip14/DHHC17 knockout mice.

Palmitoylation of neurotransmitter receptors and associated scaffold proteins regulates their membrane association in a rapid, reversible, and activity-dependent fashion. This makes palmitoylation an attractive candidate as a key regulator of the fast, reversible, and activity-dependent insertion of synaptic proteins required during the induction and expression of long-term plasticity. Here we describe that the constitutive loss of huntingtin interacting protein 14 (Hip14, also known as DHHC17), a single member of the broad palmitoyl acyltransferase (PAT) family, produces marked alterations in synaptic function in varied brain regions and significantly impairs hippocampal memory and synaptic plasticity. The data presented suggest that, even though the substrate pool is overlapping for the 23 known PAT family members, the function of a single PAT has marked effects upon physiology and cognition. Moreover, an improved understanding of the role of PATs in synaptic modification and maintenance highlights a potential strategy for intervention against early cognitive impairments in neurodegenerative disease.

Comprehensive clinical imaging, histopathological analysis and liquid biopsy-based surveillance of human uveal melanoma in a prolonged rabbit xenograft model.

Uveal melanoma is the most common intraocular tumor in adults. Our group has previously developed a human uveal melanoma animal model; however, adverse effects caused by the immunosuppressive agent, cyclosporine A, prevented animals from surviving more than 12 weeks. In this study, we tested multiple cyclosporine A doses over an extended disease course up to 20 weeks, providing complete clinical imaging of intraocular tumors, histopathological analysis and liquid biopsy biomarker analysis. Twenty albino rabbits were divided into four groups with different daily cyclosporine A schedules (0-10 mg/kg) and inoculated with human uveal melanoma cell lines, 92.1 or MP41, into the suprachoroidal space. Rabbits were monitored with fundoscopy, ultrasound and optical coherence tomography. Intraocular tumors (macroscopic or microscopic) were detected in all study animals. Tumor size and growth were correlated to cyclosporine A dose, with tumors regressing when cyclosporine A was arrested. All tumors expressed HMB-45 and MelanA; however, tumor size, pigmentation and cell morphology differed in 92.1 vs. MP41 tumors. Finally, across all groups, circulating tumor DNA from plasma and aqueous humor was detected earlier than tumor detection by imaging and correlated to tumor growth. In conclusion, using three clinically relevant imaging modalities (fundoscopy, ultrasonography and optical coherence tomography) and liquid biopsy, we were successfully able to monitor tumor progression in our rabbit xenograft model of human uveal melanoma.

Histopathological Variability and Concomitant Lesions in Pterygium in a Large Case Series.

Pterygium is a common lesion consisting of fleshy conjunctival growth extending towards the cornea. There is no documented risk of malignant transformation; however, concomitant disease is not rare, and its link to sunlight exposure indicates a risk of other malignancies. The purpose of our study is to describe histopathological features of resected pterygiums and to recognize patients at risk of other conjunctival diseases. One hundred and forty-nine formalin-fixed and paraffin-embedded pterygium samples were subjected to histopathological analysis. Histological H&E sections were obtained and digitalized using a Zeiss Axio Scan.Z1 slide scanner. Thirteen predefined morphological features were used to record histopathological changes in the epithelium and substantia propria. Neovascularization was observed in 54% of the samples. Sun damage, comprising solar elastosis and stromal plaque, was present in 81% of the samples. Variation in epithelial thickness was the most common change, with acanthosis and atrophy being observed in 62% and 26% of the samples, respectively. In our series, 21% (31/149) of pterygiums showed mild to moderate dysplasia, a finding that may be associated to ocular surface squamous neoplasia (OSSN). Moreover, 32% (47/149) of the cases showed melanocytic hyperplasia, which could represent primary acquired melanosis (PAM). There is a positive correlation between dysplasia and chronic inflammation (p=0.012) and an inverse correlation with epithelial atrophy (p=0.001) and neovascularization (p=0.05). Similarly, a positive correlation is observed between goblet cell hyperplasia and melanocytic hyperplasia (p=0.02). Our findings show that pterygiums harbour histological features that may be suggestive of OSSN or PAM in 53% of our patients. Whilst being on the benign side of the spectrum, these two entities are known for their potential progression to malignancy. A recommendation is made for all surgically excised pterygiums to be sent for histopathological diagnosis, and clear guidelines for reporting of these lesions should be established. Associated histopathological findings suggestive of other concomitant diseases should be identified to insure adequate follow-up of these patients.

Optical Coherence Tomography of Peri-Ocular Skin Cancers: An Optical Biopsy.

INTRODUCTION: Optical coherence tomography (OCT) imaging has been used as a diagnostic tool for retinal disease for several years, and OCT apparatuses are becoming increasingly powerful. However, OCT has yet to reach its full potential in ophthalmology clinics. Alike retinal layers, it has been shown that OCT is able to generate cross-sectional images of the skin and allows visualization of skin lesions in a histopathology-like manner. OBJECTIVE: We aim to validate OCT as an imaging modality for peri-ocular skin cancer. Through a series of cases, we highlight findings for 3 common eyelid malignancies: basal cell carcinoma, squamous cell carcinoma and sebaceous carcinoma. We propose an OCT image-based signature for basal cell carcinoma. METHODS: This is a prospective study. Fifty-eight lesions suspicious of malignancy from 57 patients were subjected to OCT imaging prior to the surgical excision of the lesion. OCT images were analysed and scored according to previously identified OCT features. Eight representative examples are presented, highlighting the OCT patterns for each malignancy side by side to its corresponding histopathological sections. RESULTS: Out of the 58 lesions analysed, 53 were malignant. A loss of the dermal-epidermal junction is observed in all malignant lesions. A strong link is observed between the presence of subepithelial hyporeflective nests on OCT and the diagnosis of basal cell carcinoma (present in 83% of cases). Conversely, lesions of epithelial origin such as squamous cell carcinoma are most often represented on OCT by acanthosis. Two supplementary cases, one basal cell carcinoma and one sebaceous carcinoma, are provided to illustrate how OCT imaging is a valuable tool in cases where clinical observations may be unusual. CONCLUSIONS: We provide evidence supporting the use of OCT for the evaluation of peri-ocular cancers. OCT enables visualization of the skin layers in vivo, before biopsy. Our results show that certain OCT features can contribute to include or exclude a diagnosis of basal cell carcinoma. By integrating this non-invasive imaging methodology into the routine assessment of peri-ocular skin lesions, especially in health care centres where access to specialists is limited, OCT imaging can increase clinical precision, reduce delays in patient referral and enhance patient care.

Intratumoral Heterogeneity in Uveal Melanoma.

Tumor biopsies in uveal melanoma (UM) serve mainly the purpose of prognostication and assessment of individual metastatic risk, but can be used for diagnosis in selected cases. The importance of precise information is paramount for selecting adequate surveillance protocols, patient counseling, and optimization of treatment strategies. However, intratumoral heterogeneity and sample representativity are major concerns and can interfere with the correct prediction of the patient's prognosis. We report a series of cases of UM with distinct morphologically identifiable areas, highlighting the differences in clinical behavior, as well as histopathological and genetic features.

Cardiorespiratory Response during the 1-min Sit-to-Stand Test in Chronic Obstructive Pulmonary Disease.

PURPOSE: This study aimed to assess the cardiorespiratory response during a 1-min sit-to-stand test (1STS) in comparison with cycling cardiopulmonary exercise test (CPET) in people with chronic obstructive pulmonary disease (COPD) and in healthy subjects and to evaluate whether 1STS may induce leg fatigue in these individuals. METHODS: Fourteen people with severe COPD and 12 healthy subjects performed a 1STS and a CPET during which cardiorespiratory response, perception of dyspnea, and leg fatigue were assessed. Quadriceps strength was assessed before and after 1STS, and contractile fatigue was defined as a postexercise fall in quadriceps twitch force greater than 15% of resting values. RESULTS: In COPD, peak V˙O2, V˙E, and HR achieved during 1STS reached 113%, 103%, and 93% of the corresponding values during CPET, respectively. Decrease in SpO2 from preexercise to peak exercise and the magnitude of dynamic hyperinflation were similar between 1STS and CPET. Borg dyspnea and leg fatigue scores were higher for CPET than 1STS. In healthy subjects, peak cardiorespiratory demand and symptom scores were higher during CPET compared with 1STS. A V˙O2 overshoot during recovery was observed only in people with COPD. After 1STS, the V˙O2 half-time recovery of COPD was 152 ± 25 s compared with 74 ± 18 in healthy subjects (P < 0.01). Ten people with COPD and five healthy subjects were considered as fatiguers. CONCLUSION: The 1STS induced a similar cardiorespiratory stress to that of CPET and was associated with contractile quadriceps fatigue in people with severe COPD. The V˙O2 overshoot and slower recovery time of cardiorespiratory variables seen in COPD demonstrate the clinical relevance of monitoring the recovery phase of exercise.

An unusual orbital tumor in an adult: Granuloma annulare.

PURPOSE: Granuloma annulare (GA) is a rare clinical entity that does not classically arise from the peri-orbital area in adults. The purpose of this case report is to present a 69-year-old female with GA of the orbit. As well, the pathological and immunohistochemical features of these tumors will be discussed. OBSERVATIONS: One case of GA of the orbit was identified from a tertiary ophthalmology referral centre. Clinical and histopathological features of the case were reviewed. Other cases of GA were also retrieved from the literature and addressed in this report. CONCLUSION AND IMPORTANCE: Granuloma annulare is a rare orbital lesion in adults. It is known to typically arise on the hands and feet of children. This lesion must be distinguished from necrobiotic xanthogranuloma (NXG), which is a progressive peri-orbital dermatosis seen in middle age men and women. GA is thought to be a benign, often self-resolving condition, whereas NXG tends to be linked to other systemic conditions and may have a poorer prognosis.Differentiating this rare orbital tumor from necrobitotic xanthogranuloma (NXG) is essential, as both a systemic work-up and follow-up must be appropriately arranged. A comprehensive description of pathognomonic microscopic features of GA and NXG is reviewed to achieve the correct diagnosis.

Novel application of anterior segment optical coherence tomography for periocular imaging.

OBJECTIVE: This study aims to assess the use of optical coherence tomography (OCT) imaging for periocular skin lesions and to determine which characteristic features of these images can be correlated to histopathology. DESIGN: This is an ongoing prospective study with Research Ethics Board approval. PARTICIPANTS: Fifty patients over 18 years old with lesions clinically suspicious of nonmelanoma skin cancer on the periocular region were included in this study. METHODS: After consent was obtained, clinical photographs and dermatoscopic images were obtained (DermLite II Hybrid M) from the lesion and its contralateral side. Subsequently, the patient was subjected to OCT imaging using the anterior segment module of a spectral domain OCT (Optovue Avanti) and images of the contralateral skin were also obtained. Surgical excision of the lesion was performed and sent for histopathological examination as per routine treatment. OCT images were then correlated to their matching digitalized histopathology section (Philips Ultra Fast Scanner 1.6 RA). RESULTS: Based on the OCT images acquired from 50 patients, 8 predominant architectural features have been correlated to histopathology: hyperkeratosis, acanthosis, loss of dermal-epidermal junction delineation, hyporeflective tumour nests, cystic structures, "bunch of grapes" nodules, hyperreflective nests, and ulcerations. Results observed from 45 malignant lesions (basal cell carcinoma, squamous cell carcinoma, and sebaceous gland carcinoma) suggest that groups of features and their layout within the same OCT image may be associated to specific tumour characteristics. CONCLUSIONS: Current data suggest that anterior-segment OCT imaging is a noninvasive imaging modality for periocular lesions and may be a valuable tool to help differentiate between some tumour types before a biopsy is performed.

Optimizing optical coherence tomography and histopathology correlation in retinal imaging.

OBJECTIVE: To develop a methodology to correlate optical coherence tomography (OCT) images and histopathological sections from the same eye. Part 1: To determine the best fixative for optimal OCT and histopathological analysis in post-mortem eyes. Part 2: A protocol is proposed to correlate histopathological features and OCT scans from the same post-mortem eyes. DESIGN: Experimental study. PARTICIPANTS: Part 1: Twenty-three rabbit eyes and 14 post-mortem human eyes. Part 2: Nineteen post-mortem human eyes. METHODS: Part 1: Six different fixatives were tested, and specimens were evaluated on 4 criteria: globe shape, structure opacification, retinal detachment, and nuclear details. Part 2: Based on the findings from Part 1, fixed human eyes were imaged using OCT. Orientation-controlled histopathological processing was performed to obtain serial tissue sections from paraffin embedded tissue, which were matched to corresponding OCT images. RESULTS: Part 1: Of the 6 fixatives, 2% glutaraldehyde and Davidson's solution met the proposed criteria in rabbit eyes. Of these, glutaraldehyde showed similar results in human eyes and was selected for Part 2. Part 2: Using anatomical landmarks, cross-sectional histopathological sections in the same orientation as the OCT images were correlated to their corresponding OCT images. Retinal lesions such as a macular hole, an epiretinal membrane, and the presence of drusen were easily correlated, proving the reliability of our methodology. Moreover, the photoreceptor's inner/outer junction was correlated to a hyperreflective band on OCT. CONCLUSIONS: A standardized protocol was developed to correlate OCT images and histopathological findings by generating serial cross-sections of the retina, which can be used to better understand otherwise ambiguous OCT findings.

The Use of Digital Microscopy to Compare the Thicknesses of Normal Corneas and Ex Vivo Rejected Corneal Grafts with a Focus on the Descemet's Membrane.

OBJECTIVE: To compare the thickness of corneal layers, specifically the Descemet's membrane (DM), in normal corneas and in failed grafts due to rejection (FGRs) using the digital histopathology and to propose a model for the measurement of corneal layers using this method. METHODS: This is a prospective, cross-sectional study performed at the MUHC-McGill University Ocular Pathology & Translational Research Laboratory (McGill University, Montreal, Canada). Histopathological sections of 25 normal human corneas and 40 FGRs were fully digitalized and examined. Inclusion criteria: samples diagnosed as normal corneas or FGRs, from patients older than 18 years of age. Exclusion criteria: histopathological sections without adequate tissue or missing epidemiological information. For each sample, the thicknesses of the epithelium, stroma, and DM were acquired. From a perpendicular plane of reference, two central measurements and two nasal and two temporal peripheral measurements were obtained. RESULTS: There were differences between the normal and FGR groups in the mean central thickness of the epithelium (p < 0.001), the nasal and temporal stromal regions (p < 0.001), and of the DM in the nasal and temporal regions (p < 0.001). Compared with the extremities of the sample (nasal and temporal), the mean thickness of the DM in normal corneas was lower in the central region (p < 0.001), and this difference was not found in the FGR group. CONCLUSIONS: Normal corneas have a thinner epithelium in the central region than the FGR group. In addition, the stroma and DM thicknesses of the nasal and temporal periphery were significantly higher in normal corneas than in those from the FGR group. The digital microscopy protocol applied in this study may be useful for further research studies regarding cornea and other tissues.

Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice.

LRRK2 mutations produce end-stage Parkinson's disease (PD) with reduced nigrostriatal dopamine, whereas, asymptomatic carriers have increased dopamine turnover and altered brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and mitochondrial abnormalities occur in aged G2019S mutant knock-in (GKI) mice. Conversely, cultured GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic glutamate and dopamine function across a range of ages. Young GKI mice exhibit more vertical exploration, elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These phenomena decline with age, but are stable in littermates. In young GKI mice, dopamine transients are slower, independent of dopamine transporter (DAT), increasing the lifetime of extracellular dopamine. Slowing of dopamine transients is observed with age in littermates, suggesting premature ageing of dopamine synapses in GKI mice. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes, making them amenable to investigation of early pathophysiological, and later parkinsonian-like, alterations. This model will prove valuable in efforts to develop neuroprotection for PD.

Chronic and acute LRRK2 silencing has no long-term behavioral effects, whereas wild-type and mutant LRRK2 overexpression induce motor and cognitive deficits and altered regulation of dopamine release.

INTRODUCTION: Germline silencing of the PD-related protein LRRK2 does not alter glutamate or dopamine release in adult mice, but some exploratory abnormalities have been reported with ageing. Contrastingly, high levels of human LRRK2 cause locomotor alterations and cognitive deficits accompanied by reduced striatal dopamine levels, with the latter also observed in G2019S mutant mice. Comparative cognitive and motor behavioral testing of LRRK2 KO, overexpressor and mutant overexpressor mice has not previously been reported. METHODS: Parallel, comparative behavioral characterization was performed assessing motor and cognitive abilities. Striatal antisense oligonucleotide injections were conducted to investigate the effects of acute LRRK2 silencing on behavior and dopamine fiber density. Striatal synaptosomes prepared from hG2019S mice assessed vesicular release of dopamine and its sensitivity to D2 autoreceptor stimulation. RESULTS: Genetic ablation of LRRK2 has no long-term consequences on motor or cognitive function. Consistently, no effects on behavior or dopaminergic fiber density were observed following acute striatal silencing. Conversely, 12-month OE mice show persistent locomotor deficits and worsening of cognitive abilities; whereas, hG2019S mice display early hyperactivity and effective learning and memory that progress to decreased motor and cognitive deficits at older ages. The G2019S mutation does not affect vesicular dopamine release, but decreases its sensitivity to D2-mediated inhibition. CONCLUSION: LRRK2 silencing is well tolerated in mouse, arguing PD does not result from LRRK2 loss of function. High levels of WT and G2019S LRRK2 produce similar but temporally distinct phenotypes, potentially modeling different stages of disease progression. The data implicate gain of LRRK2 function in the pathogenesis of PD.

Behavioral deficits and striatal DA signaling in LRRK2 p.G2019S transgenic rats: a multimodal investigation including PET neuroimaging.

BACKGROUND: A major risk-factor for developing Parkinson's disease (PD) is genetic variability in leucine-rich repeat kinase 2 (LRRK2), most notably the p.G2019S mutation. Examination of the effects of this mutation is necessary to determine the etiology of PD and to guide therapeutic development. OBJECTIVE: Assess the behavioral consequences of LRRK2 p.G2019S overexpression in transgenic rats as they age and test the functional integrity of the nigro-striatal dopamine system. Conduct positron emission tomography (PET) neuroimaging to compare transgenic rats with previous data from human LRRK2 mutation carriers. METHODS: Rats overexpressing human LRRK2 p.G2019S were generated by BAC transgenesis and compared to non-transgenic (NT) littermates. Motor skill tests were performed at 3, 6 and 12 months-of-age. PET, performed at 12 months, assessed the density of dopamine and vesicular monoamine transporters (DAT and VMAT2, respectively) and measured dopamine synthesis, storage and availability. Brain tissue was assayed for D2, DAT, dopamine and cAMP-regulated phosphoprotein (DARPP32) and tyrosine hydroxylase (TH) expression by Western blot, and TH by immunohistochemistry. RESULTS: Transgenic rats had no abnormalities in measures of striatal dopamine function at 12 months. A behavioral phenotype was present, with LRRK2 p.G2019S rats performing significantly worse on the rotarod than non-transgenic littermates (26% reduction in average running duration at 6 months), but with normal performance in other motor tests. CONCLUSIONS: Neuroimaging using dopaminergic PET did not recapitulate prior studies in human LRRK2 mutation carriers. Consistently, LRRK2 p.G2019S rats do not develop overt neurodegeneration; however, they do exhibit behavioral abnormalities.

Blockade of ventral midbrain NMDA receptors enhances brain stimulation reward: a preferential role for GluN2A subunits.

Ventral midbrain (VM) neurons that project to limbic structures play a role in reward and incentive motivation. It has been suggested that a reward-related signal is transmitted when the firing rate of VM dopamine neurons shifts from a tonic to a phasic mode. Since glutamate is necessary for this transduction process, it is likely to play a role in reward signaling. This study was aimed at determining the effect of VM N-Methyl-D-Aspartate (NMDA) receptor blockade on reward induced by electrical brain stimulation. Experiments were performed on rats trained to self-administer an electrical stimulation in the medial posterior mesencephalon. Reward thresholds were measured with the curve-shift paradigm before and after bilateral VM injections of the following NMDA receptor antagonists: R-CPP, 3-(R-2-Carboxypiperazin-4-yl)-propyl-1 phosphonic acid, (0, 20.6, 41.2 and 82.5 pmol/0.5 µl/side), PPPA, (2R,4S)-4-(3-Phosphonopropyl)-2-piperidinecarboxylic acid, (0, 0.825 and 1.65 nmol/0.5 µl/side) orRo04-5595, 1-[2-(4-Chlorophenyl)ethyl]-1,2,3,4-tetrqahydro-6-methoxy-2-methyl-7-isoquinolinol hydrochloride (0, 0.825, 1.65 nmol/0.5 µl/side). R-CPP and PPPA produced a dose and time dependent decrease in reward threshold, an effect that was, at some doses and times after the injection, accompanied by an increase in maximum responses. These effects were not observed with Ro04-5595 over the range of doses tested. While previous studies suggest a role for glutamate in reward signaling, the present results show that VM glutamate exerts a tonic inhibition on the reward-relevant pathway. The selectivity of Ro04-5595 for NMDA receptors composed of GluN2B subunits and the higher affinity of R-CPP and PPPA for GluN2A suggest that the inhibition is mediated by receptors composed of GluN2A subunits.