Katrina L. Paumier

TRANSFER OF HOST-DERIVED ALPHA SYNUCLEIN TO GRAFTED DOPAMINERGIC NEURONS IN RAT

Multiple laboratories have recently demonstrated that long-term dopaminergic transplants form Lewy bodies in patients with Parkinsons disease. Debate has arisen as to whether these Lewy bodies form from the transfer of α synuclein from the host to the graft or whether they form from intrinsic responses of the graft from being placed into what was, or became, an inflammatory focus. To test whether the former hypothesis was possible, we grafted fetal rat ventral mesencephalon into the dopamine depleted striatum of rats that had previously received 6-hydroxydopamine lesions. One month after the transplant, rats received viral over expression of human α synuclein (AAV2/6-α synuclein) or green fluorescent protein (AAV2/6-GFP) into the striatum rostral to the grafts. Care was taken to make sure that the AAV injections were sufficiently distal to the graft so no cells would be directly transfected. All rats were sacrificed five weeks after the virus injections. Double label immunohistochemistry combined with confocal microscopy revealed that a small number of grafted tyrosine hydroxylase (TH) neurons (5.7% ± 1.5% (mean ± SEM) of grafted dopamine cells) expressed host derived α synuclein but none of the grafted cells expressed host-derived GFP. The α synuclein in a few of these cells was misfolded and failed to be digested with proteinase K. These data indicate that it is possible for host derived α synuclein to transfer to grafted neurons supporting the concept that this is one possible mechanism by which grafted dopamine neurons form Lewy bodies in Parkinsons disease patients.

Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinsons disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague-Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting that PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time-point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.

Stimulation of the Rat Subthalamic Nucleus is Neuroprotective Following Significant Nigral Dopamine Neuron Loss

Deep brain stimulation of the subthalamic nucleus (STN-DBS) is efficacious in treating the motor symptoms of Parkinsons disease (PD). However, the impact of STN-DBS on the progression of PD is unknown. Previous preclinical studies have demonstrated that STN-DBS can attenuate the degeneration of a relatively intact nigrostriatal system from dopamine (DA)-depleting neurotoxins. The present study examined whether STN-DBS can provide neuroprotection in the face of prior significant nigral DA neuron loss similar to PD patients at the time of diagnosis. STN-DBS between 2 and 4 weeks after intrastriatal 6-hydroxydopamine (6-OHDA) provided significant sparing of DA neurons in the SN of rats. This effect was not due to inadvertent lesioning of the STN and was dependent upon proper electrode placement. Since STN-DBS appears to have significant neuroprotective properties, initiation of STN-DBS earlier in the course of PD may provide added neuroprotective benefits in addition to its ability to provide symptomatic relief.

Transplantation of subventricular zone neural precursors induces an endogenous precursor cell response in a rat model of Parkinson's disease.

Realistically, future stem cell therapies for neurological conditions including Parkinsons disease (PD) will most probably entail combination treatment strategies, involving both the stimulation of endogenous cells and transplantation. Therefore, this study investigates these two modes of neural precursor cell (NPC) therapy in concert in order to determine their interrelationships in a rat PD model. Human placental alkaline phosphatase (hPAP)‐labeled NPCs were transplanted unilaterally into host rats which were subsequently infused ipsilaterally with 6‐hydroxydopamine (6‐OHDA). The reaction of host NPCs to the transplantation and 6‐OHDA was tracked by bromodeoxyuridine (BrdU) labeling. Two weeks after transplantation, in animals transplanted with NPCs we found evidence of elevated host subventricular zone NPC proliferation, neurogenesis, and migration to the graft site. In these animals, we also observed a significant preservation of striatal tyrosine hydroxylase (TH) expression and substantia nigra TH cell number. We have seen no evidence that neuroprotection is a product of dopamine neuron replacement by NPC‐derived cells. Rather, the NPCs expressed glial cell line‐derived neurotrophic factor (GDNF), sonic hedgehog (Shh), and stromal cell‐derived factor 1 alpha (SDF1α), providing a molecular basis for the observed neuroprotection and endogenous NPC response to transplantation. In summary, our data suggests plausible synergy between exogenous and endogenous NPC actions, and that NPC implantation before the 6‐OHDA insult can create a host microenvironment conducive to stimulation of endogenous NPCs and protection of mature nigral neurons. J. Comp. Neurol. 515:102–115, 2009.

The DIAN-TU Next Generation Alzheimer's prevention trial: Adaptive design and disease progression model

The Dominantly Inherited Alzheimer Network Trials Unit (DIAN‐TU) trial is an adaptive platform trial testing multiple drugs to slow or prevent the progression of Alzheimers disease in autosomal dominant Alzheimers disease (ADAD) families. With completion of enrollment of the first two drug arms, the DIAN‐TU now plans to add new drugs to the platform, designated as the Next Generation (NexGen) prevention trial.

Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis

Cerebrospinal fluid analysis and other measurements of amyloidosis, such as amyloid‐binding positron emission tomography studies, are limited by cost and availability. There is a need for a more practical amyloid β (Aβ) biomarker for central nervous system amyloid deposition.

Tricyclic antidepressants delay the need for dopaminergic therapy in early Parkinson's disease.

This study examined whether antidepressants delay the need for dopaminergic therapy or change the degree of motor impairment and disability in a population of early Parkinsons disease (PD) patients. Preclinical studies have indicated that antidepressants modulate signaling pathways involved in cell survival and plasticity, suggesting they may serve to both treat PD‐associated depression and slow disease progression. A patient‐level meta‐analysis included 2064 patients from the treatment and placebo arms of the following trials: FS1, FS‐TOO, ELLDOPA, QE2, TEMPO, and PRECEPT. Depression severity was determined at baseline, and antidepressant use was reported in a medication log each visit. Kaplan–Meier curves and time‐dependent Cox proportional hazards models determined associations between depression severity and antidepressant use with the primary outcome, time to initiation of dopaminergic therapy. ANCOVAs determined associations with the secondary outcome, degree of motor impairment and disability, reported as annualized change in UPDRS scores from baseline to final visit. When controlling for baseline depression, the initiation of dopaminergic therapy was delayed for subjects taking tricyclic antidepressants compared with those not taking antidepressants. No significant differences were found in UPDRS scores for subjects taking antidepressants compared with those not taking antidepressants. Tricyclic antidepressants are associated with a delay in reaching the end point of need to start dopaminergic therapy. The lack of change in overall UPDRS scores suggests the delay was not attributable to symptomatic effects.

INCREASED CELL SUSPENSION CONCENTRATION AUGMENTS THE SURVIVAL RATE OF GRAFTED TYROSINE HYDROXYLASE IMMUNOREACTIVE NEURONS

The poor survival rate (5-20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinsons disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/microl and transplanted into two 0.5 microl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl groups (p=0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.

Tricyclic antidepressant treatment evokes regional changes in neurotrophic factors over time within the intact and degenerating nigrostriatal system.

In addition to alleviating depression, trophic responses produced by antidepressants may regulate neural plasticity in the diseased brain, which not only provides symptomatic benefit but also potentially slows the rate of disease progression in Parkinsons disease (PD). Recent in vitro and in vivo data provide evidence that neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) may be key mediators of the therapeutic response to antidepressants. As such, we conducted a cross-sectional time-course study to determine whether antidepressant-mediated changes in neurotrophic factors occur in relevant brain regions in response to amitriptyline (AMI) treatment before and after intrastriatal 6-hydroxydopamine (6OHDA). Adult male Wistar rats were divided into seven cohorts and given daily injections (i.p.) of AMI (5mg/kg) or saline throughout the duration of the study. In parallel, various cohorts of intact or parkinsonian animals were sacrificed at specific time points to determine the impact of AMI treatment on trophic factor levels in the intact and degenerating nigrostriatal system. The left and right hemispheres of the substantia nigra, striatum, frontal cortex, piriform cortex, hippocampus, and anterior cingulate cortex were dissected, and BDNF and GDNF levels were measured with ELISA. Results show that chronic AMI treatment elicits effects in multiple brain regions and differentially regulates levels of BDNF and GDNF depending on the region. Additionally, AMI halts the progressive degeneration of dopamine (DA) neurons elicited by an intrastriatal 6-OHDA lesion. Taken together, these results suggest that AMI treatment elicits significant trophic changes important to DA neuron survival within both the intact and degenerating nigrostriatal system.

Spatial patterns of neuroimaging biomarker change in individuals from families with autosomal dominant Alzheimer's disease: a longitudinal study

Background Models of Alzheimer disease propose a sequence of amyloid-β (Aβ) accumulation, hypometabolism, and structural declines that precede the onset of clinical dementia. These pathological features evolve both temporally and spatially in the brain. This study aimed to characterize where in the brain and when in the course of the disease neuroimaging biomarkers become abnormal. Methods We analyzed data from mutation non-carriers, asymptomatic carriers, and symptomatic carriers collected between January 1st 2009 and December 31st 2015 from families carrying PSEN1, PSEN2, or APP mutations enrolled in the Dominantly Inherited Alzheimer’s Network. We analyzed [11C]Pittsburgh Compound B positron emission tomography (PiB PET), [18F]Fluorodeoxyglucose (FDG PET), and structural magnetic resonance imaging (MRI) data using regions of interest to assess change throughout the brain. We estimated rates of biomarker change as a function of estimated years from symptom onset at baseline using linear mixed-effects models and determined the earliest point at which biomarker trajectories differed between mutation carriers and non-carriers. Findings PiB PET was available for 346 individuals, with 162 having longitudinal imaging; FDG PET was available for 352 (175 longitudinal); and MRI data was available for 377 (201 longitudinal). We found a sequence to pathological changes, with rates of Aβ deposition in mutation carriers being significantly different from non-carriers first (on average across regions that showed a significant difference at −18·9 (sd 3·3) years before expected onset), followed by hypometabolism (−14·1 years, sd 5·1) and lastly structural declines (−4·7 years, sd 4·2). This biomarker ordering was preserved in most, but not all, regions. The temporal emergence within a biomarker varied across the brain, with the precuneus being the first cortical region in each modality to show divergence between groups (−22·2 years before expected onset for Aβ accumulation, −18·8 years for hypometabolism, and −13·0 years for cortical thinning). Interpretation Mutation carriers had elevations in Aβ deposition, reduced glucose metabolism, and cortical thinning which preceded the expected onset of dementia. Accrual of these pathologies varied throughout the brain, suggesting differential regional and temporal vulnerabilities to Aβ, metabolic decline, and structural atrophy, which should be taken into account when using biomarkers in a clinical setting as well as designing and evaluating clinical trials.