Venette Inskeep

Reversal of advanced disease in lysosomal acid lipase deficient mice: A model for lysosomal acid lipase deficiency disease

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TG) and cholesteryl esters (CE) in lysosomes. Mutations of the LIPA gene lead to Wolman disease (WD) and cholesterol ester storage disease (CESD). The disease hallmarks include hepatosplenomegaly and extensive storage of CE and/or TG. The effects of intravenous investigational enzyme therapy (ET) on survival and efficacy were evaluated in Lipa knock out, lal-/- mice with advanced disease using recombinant human LAL (rhLAL). Comparative ET was conducted with lower doses (weekly, 0.8 and 3.2mg/kg) beginning at 16 weeks (study 1), and with higher dose (10mg/kg) in early (8-weeks), middle (16-weeks) and late (24-weeks) disease stages (study 2). In study 1, rhLAL extended the life span of lal-/- mice in a dose dependent manner by 52 (0.8 mg/kg) or 94 (3.2mg/kg) days. This was accompanied by partial correction of cholesterol and TG levels in spleen and liver. In study 2, the high dose resulted in a significant improvement in organ size (liver, spleen and small intestine) and tissue histology as well as significant decreases in cholesterol and TG in all three groups. In the treated livers and spleens the cholesterol and TG levels were reduced to below treatment initiation levels indicating a reversal of disease manifestations, even in advanced disease. ET diminished liver fibrosis and macrophage proliferation. These results show that LAL deficiency can be improved biochemically and histopathologically by various dosages of ET, even in advanced disease.

Prosaposin is a regulator of progranulin levels and oligomerization

Progranulin (GRN) loss-of-function mutations leading to progranulin protein (PGRN) haploinsufficiency are prevalent genetic causes of frontotemporal dementia. Reports also indicated PGRN-mediated neuroprotection in models of Alzheimers and Parkinsons disease; thus, increasing PGRN levels is a promising therapeutic for multiple disorders. To uncover novel PGRN regulators, we linked whole-genome sequence data from 920 individuals with plasma PGRN levels and identified the prosaposin (PSAP) locus as a new locus significantly associated with plasma PGRN levels. Here we show that both PSAP reduction and overexpression lead to significantly elevated extracellular PGRN levels. Intriguingly, PSAP knockdown increases PGRN monomers, whereas PSAP overexpression increases PGRN oligomers, partly through a protein–protein interaction. PSAP-induced changes in PGRN levels and oligomerization replicate in human-derived fibroblasts obtained from a GRN mutation carrier, further supporting PSAP as a potential PGRN-related therapeutic target. Future studies should focus on addressing the relevance and cellular mechanism by which PGRN oligomeric species provide neuroprotection.

Neuronopathic Gaucher disease: dysregulated mRNAs and miRNAs in brain pathogenesis and effects of pharmacologic chaperone treatment in a mouse model

Defective lysosomal acid β-glucosidase (GCase) in Gaucher disease causes accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) that distress cellular functions. To study novel pathological mechanisms in neuronopathic Gaucher disease (nGD), a mouse model (4L;C*), an analogue to subacute human nGD, was investigated for global profiles of differentially expressed brain mRNAs (DEGs) and miRNAs (DEmiRs). 4L;C* mice displayed accumulation of GC and GS, activated microglial cells, reduced number of neurons and aberrant mitochondrial function in the brain followed by deterioration in motor function. DEGs and DEmiRs were characterized from sequencing of mRNA and miRNA from cerebral cortex, brain stem, midbrain and cerebellum of 4L;C* mice. Gene ontology enrichment and pathway analysis showed preferential mitochondrial dysfunction in midbrain and uniform inflammatory response and identified novel pathways, axonal guidance signaling, synaptic transmission, eIF2 and mammalian target of rapamycin (mTOR) signaling potentially involved in nGD. Similar analyses were performed with mice treated with isofagomine (IFG), a pharmacologic chaperone for GCase. IFG treatment did not alter the GS and GC accumulation significantly but attenuated the progression of the disease and altered numerous DEmiRs and target DEGs to their respective normal levels in inflammation, mitochondrial function and axonal guidance pathways, suggesting its regulation on miRNA and the associated mRNA that underlie the neurodegeneration in nGD. These analyses demonstrate that the neurodegenerative phenotype in 4L;C* mice was associated with dysregulation of brain mRNAs and miRNAs in axonal guidance, synaptic plasticity, mitochondria function, eIF2 and mTOR signaling and inflammation and provides new insights for the nGD pathological mechanism.

Modulating ryanodine receptors with dantrolene attenuates neuronopathic phenotype in Gaucher disease mice.

Neuronopathic Gaucher disease (nGD) manifests as severe neurological symptoms in patients with no effective treatment available. Ryanodine receptors (Ryrs) are a family of calcium release channels on intracellular stores. The goal of this study is to determine if Ryrs are potential targets for nGD treatment. A nGD cell model (CBE-N2a) was created by inhibiting acid β-glucosidase (GCase) in N2a cells with conduritol B epoxide (CBE). Enhanced cytosolic calcium in CBE-N2a cells was blocked by either ryanodine or dantrolene, antagonists of Ryrs and by Genz-161, a glucosylceramide synthase inhibitor, suggesting substrate-mediated ER-calcium efflux occurs through ryanodine receptors. In the brain of a nGD (4L;C*) mouse model, expression of Ryrs was normal at 13 days of age, but significantly decreased below the wild type level in end-stage 4L;C* brains at 40 days. Treatment with dantrolene in 4L;C* mice starting at postnatal day 5 delayed neurological pathology and prolonged survival. Compared to untreated 4L;C* mice, dantrolene treatment significantly improved gait, reduced LC3-II levels, improved mitochondrial ATP production and reduced inflammation in the brain. Dantrolene treatment partially normalized Ryr expression and its potential regulators, CAMK IV and calmodulin. Furthermore, dantrolene treatment increased residual mutant GCase activity in 4L;C* brains. These data demonstrate that modulating Ryrs has neuroprotective effects in nGD through mechanisms that protect the mitochondria, autophagy, Ryr expression and enhance GCase activity. This study suggests that calcium signalling stabilization, e.g. with dantrolene, could be a potential disease modifying therapy for nGD.

Progression of Behavioral and CNS Deficits in a Viable Murine Model of Chronic Neuronopathic Gaucher Disease.

To study the neuronal deficits in neuronopathic Gaucher Disease (nGD), the chronological behavioral profiles and the age of onset of brain abnormalities were characterized in a chronic nGD mouse model (9V/null). Progressive accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) in the brain of 9V/null mice were observed at as early as 6 and 3 months of age for GC and GS, respectively. Abnormal accumulation of α-synuclein was present in the 9V/null brain as detected by immunofluorescence and Western blot analysis. In a repeated open-field test, the 9V/null mice (9 months and older) displayed significantly less environmental habituation and spent more time exploring the open-field than age-matched WT group, indicating the onset of short-term spatial memory deficits. In the marble burying test, the 9V/null group had a shorter latency to initiate burying activity at 3 months of age, whereas the latency increased significantly at ≥12 months of age; 9V/null females buried significantly more marbles to completion than the WT group, suggesting an abnormal response to the instinctive behavior and an abnormal activity in non-associative anxiety-like behavior. In the conditional fear test, only the 9V/null males exhibited a significant decrease in response to contextual fear, but both genders showed less response to auditory-cued fear compared to age- and gender-matched WT at 12 months of age. These results indicate hippocampus-related emotional memory defects. Abnormal gait emerged in 9V/null mice with wider front-paw and hind-paw widths, as well as longer stride in a gender-dependent manner with different ages of onset. Significantly higher liver- and spleen-to-body weight ratios were detected in 9V/null mice with different ages of onsets. These data provide temporal evaluation of neurobehavioral dysfunctions and brain pathology in 9V/null mice that can be used for experimental designs to evaluate novel therapies for nGD.

521. Platelets Transfusion New Role as Brain Therapeutics for Acute Neuronopathic Gaucher Disease

There has no effective treatment for neuronopathic Gaucher Disease (nGD) due to the difficulty of therapeutics to cross the blood-brain-barrier. Platelets are blood elements that contain cytoplasmic secretory vesicles containing proteins involved in hemostasis, inflammation and angiogenesis. We have recently shown that platelets/megakaryocytes could serve as efficient and protective depots for lysosomal enzyme generation and distribution. In this study the potential therapeutic benefits of platelet transfusion in treating neuronopathic GD (nGD) was evaluated using a murine model of acute nGD (4L;C), which resembles types 2 and 3 Gaucher disease. Platelets containing significant amounts of wild type acid s-glucosidase enzyme (GCase) were isolated from GFP mice and transfused weekly into 4L;C mice for four times beginning at 21 days of age, resulting in an average 10-13% of donor-derived GFP+ platelets in circulation with half-life of 52 hr. Importantly, moderate platelet transfusion led to significantly increase of life-span in 4L;C mice from 47 days to 55 days with reduction of splenomegaly. The accumulation of glycosylsphingosin was significantly reduced in spleen, liver and brain. Moreover, the abnormal walk patterns with splaying of hindlimbs exhibited in 4L;C mice were improved significantly in platelet-treated mice, indicating amelioration of motor neuron dysfunction and hindlimb paresis. The improvement of short-term memory deficit in treated 4L;C mice was evidenced in repeated open-field test. Immunohistochemistry analysis showed that the elevated brain CD68+ signals, a marker for activated microglia cells, were normalized in cerebellum folium and middle brain regions, and partially corrected in the cortex, cerebellum DCN, brainstem, thalamus and spinal cord of treated 4L;C mice, indicating the reduction of pro-inflammation by platelet transfusion. Notably, RIPK3-positive signals within Purkinje neurons in cerebellum of nGD mice were significantly reduced, suggesting therapeutic benefit of platelet transfusion on neuronal necroptosis. This study reveals an under-appreciated, unexpected role for platelets in treating CNS diseases. These results provide proof of concept that platelet transfusion can achieve CNS benefits with brain substrate reduction and improvement in neurological function, as well as reducing pro-inflammation and neuronal necroptosis. The findings will open a door for new clinical approaches with platelet transfusion in treating nGD and potentially other brain diseases.

374. Therapeutic Effect of Platelet Transfusions for Acute Neuronopathic Gaucher Disease

Enzyme replacement therapy has led to resolution of most visceral manifestations in Gaucher disease (GD), but it is also limited by the instability of acid beta -glucosidase enzyme (GCase) in circulation, liver 1st bypass, and high cost. In addition, intravenous enzyme administration has not been effective for ameliorating central nervous system (CNS) deficits. Platelets are blood elements that contain cytoplasmic secretory vesicles. The involvement of platelet containing proteins in inflammation and angiogenesis has recently been indicated. We have recently shown that platelets/megakaryocytes could serve as efficient and protective depots for lysosomal enzyme generation and distribution. In this study the potential therapeutic benefits of platelet transfusion in treating neuronopathic GD (nGD) was evaluated using a murine model of acute nGD (4L;C). This model resembles the CNS phenotype and biochemistry of types 2 and 3 Gaucher disease, and premature death results from progressive CNS disease. Platelets containing significant amounts of wild type GCase (19 U/109 plt) were isolated from GFP mice and transfused weekly for four times into 4L;C mice beginning at 21 days of age. The average percentages of donor-derived platelets were at mean of 10% to 12.9% (ranging from 7.2 to 16.1%) as measured one hour after each transfusion by flow cytometry analysis with anti-CD41 staining for GFP-positive donor platelets. Clearance curves of transfused platelets were similar in 4L;C and wild-type recipients with comparable half-life of GFP+ platelets (t1/2=50 h), suggesting the lack of an immune-response against platelet-derived normal GCase in nGD mice. Importantly, the life-span of treated 4L;C mice was significantly extended from 46 days [untreated 4L;C (n=22)] to 54 days [treated, (n=7)]. Potential CNS benefits were assessed using hindlimb gait analysis that evaluated motor function. In comparison to normal controls, the untreated 4L;C mice exhibited abnormal splaying of their hindlimbs and progressive paresis. The platelet-treated 4L;C mice improved significantly toward normal behavior and had delays in the age of onset for CNS signs. Immunohistochemistry analysis showed that numbers of CD68+ cells were decreased in the cerebellum, brainstem and cortex of treated 4L;C mice, indicating the reduction of pro-inflammation by platelet transfusion. The clearance of GFP+ platelets by liver and spleen was shown by GFP signals detected in those organs of treated 4L;C mice. Quantification of the pathogenic lipids, glucosylceramide and glucosylsphingosine, is in progress to assess potential metabolic correction in various regions of brain and visceral organs in platelet-treated nGD mice. More studies are underway to further investigate possible mechanism(s) involved. In summary, our results demonstrate that repeated, moderate levels of platelet transfusion could extend the life span of nGD mice and improve CNS deficits. abbbb