Cyclosporin promotes neurorestoration and cell replacement therapy in pre-clinical models of Parkinson's disease.

The early clinical trials using fetal ventral mesencephalic (VM) allografts in Parkinson's disease (PD) patients have shown efficacy (albeit not in all cases) and have paved the way for further development of cell replacement therapy strategies in PD. The preclinical work that led to these clinical trials used allografts of fetal VM tissue placed into 6-OHDA lesioned rats, while the patients received similar allografts under cover of immunosuppression in an α-synuclein disease state. Thus developing models that more faithfully replicate the clinical scenario would be a useful tool for the translation of such cell-based therapies to the clinic

Loss of Leucine-rich Repeat Kinase 2 (LRRK2) in Rats Leads to Progressive Abnormal Phenotypes in Peripheral Organs

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson’s disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype. In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson’s disease

Loss of Leucine-Rich Repeat Kinase 2 (LRRK2) in Rats Leads to Progressive Abnormal Phenotypes in Peripheral Organs

<div><p>The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson’s disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype.  In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080705#B1" target="_blank">1</a>] and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson’s disease.</p> </div

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system x represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system x, xCT (xCT mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT mice. A proteomic and kinomic screen of the striatum of xCT mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system x plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior

1-month old LRRK2 KO rat (Cortex, kidney).

<p>A: Hematoxylin and Eosin – no observable abnormalities; B: Lipofuscin (AFIP method) – lipofuscin positive material not observed; C: Chromotrope Aniline Blue – CAB positive staining not readily visible and similar to WT rat; D: LAMP-1 – diffuse, cytoplasmic, fine granular staining with a slight increase in intensity in proximal tubular epithelium; E: LAMP-2 - intense staining of apical cytoplasm in proximal tubular epithelium similar to WT control; F: NAGLU – fine, diffuse, granular cytoplasmic staining and apical staining in proximal tubular epithelium similar to WT control (the 1, 2 and 8 month old WT rats in the second cohort had similar baseline NAGLU staining that was slightly different (apical and diffuse cytoplasmic) from those rats in the first cohort (diffuse cytoplasmic). Scale bar = 0.5 mm</p

Accumulation of Lamellar Bodies in Type II Alveolar Cells in the lung of 16-month old LRRK2 KO rats vs. WT.

<p>(A) Graphs depicting Number of Lamellar Bodies in Type II Alveolar Cells and Area of Type II Alveolar Cells. (WT n=4, KO N=4) * - denote significance by Students <i>t</i>-test, P<0.05. Values are means <u>+</u> Standard Error of the Mean. (B) Normal Lamellar Body morphology (white arrow) in WT compared to proliferation of lamellar bodies (black arrow) in KO. N: Nucleus. Scale bar = 2 µm</p

2-month old LRRK2 KO rat (Cortex, kidney).

<p>A: Hematoxylin and Eosin – slight increase in size and variability of hyaline droplets in proximal tubular epithelium (open arrow); B: Lipofuscin (AFIP method) – Lipofuscin positive material not observed; C: Chromotrope Aniline Blue – increased size, number and variability of hyaline droplets in proximal tubular epithelium (open arrows). Hyaline droplet variability was more easily identified using CAB staining when compared to H&E stains; D: LAMP-1 – increased cytoplasmic staining intensity in proximal tubular epithelium; E: LAMP-2 – increased globular cytoplasmic staining (solid arrows) with apical cytoplasmic staining- persisting in proximal tubular epithelium; F: NAGLU – increased globular staining in proximal tubular epithelium (solid arrow). Scale bar = 0.5 mm</p

Morphological changes in the kidney of 16-month old LRRK2 KO rats vs.

<div><p><b>WT</b>. </p> <p>(A) Increased number of lysosomes and electron dense material (black arrows) in KOs compared to WT (white arrow) (B) Accumulation of lipid droplets in glomerulus of KO animals compared to WT, which have no lipid droplets. N: Nucleus. Scale bar = 2 µm</p></div

4-month old LRRK2 KO rat (Cortex, kidney).

<p>A: Hematoxylin and Eosin – brown, globular pigment (dotted arrows) and intracytoplasmic, clear vacuoles in proximal tubular epithelium with occasional hyaline droplets (open arrow); B: Lipofuscin (AFIP method) – positive dark red staining of pigment (dotted arrows) and clear vacuoles in proximal tubular epithelium; C: Chromotrope Aniline Blue – Increase in number, size and variability of hyaline droplets (red, open arrows) and clear cytoplasmic vacuoles in proximal tubular epithelium; D: LAMP-1 – Globular cytoplasmic staining in proximal tubular epithelium (solid arrows); E: LAMP-2 – increased globular cytoplasmic staining (solid arrows) with apical staining persisting in proximal tubular epithelium; F: NAGLU – increase intensity of globular cytoplasmic staining (open arrows). Scale bar = 0.5 mm</p

Accumulation of lipid droplets in hepatic cells in 16-month old LRRK2 KO rats vs. WT.

<p>(A) Graphs showing changes in number of lipid droplets and density of lipid droplets per hepatocyte. (WT n=2, KO n=4) *- denote significance by Student’s <i>t</i>-test, P<0.05. Values are means <u>+</u> Standard Error of the Mean. (B) Comparison of normal accumulation of lipid droplets (white arrow) in hepatic cells in WT to increased accumulation in hepatic cells (black arrow) in KOs. N: Nucleus. Scale bar = 2 µm</p