Moving towards effective therapeutic strategies for Neuronal Ceroid Lipofuscinosis.

The Neuronal Ceroid Lipofuscinoses (NCLs) are a family of autosomal recessive neurodegenerative disorders that annually affect 1:100,000 live births worldwide. This family of diseases results from mutations in one of 14 different genes that share common clinical and pathological etiologies. Clinically, the diseases are subcategorized into infantile, late-infantile, juvenile and adult forms based on their age of onset. Though the disease phenotypes may vary in their age and order of presentation, all typically include progressive visual deterioration and blindness, cognitive impairment, motor deficits and seizures. Pathological hallmarks of NCLs include the accumulation of storage material or ceroid in the lysosome, progressive neuronal degeneration and massive glial activation. Advances have been made in genetic diagnosis and counseling for families. However, comprehensive treatment programs that delay or halt disease progression have been elusive. Current disease management is primarily targeted at controlling the symptoms rather than curing the disease. Recognizing the growing need for transparency and synergistic efforts to move the field forward, this review will provide an overview of the therapeutic approaches currently being pursued in preclinical and clinical trials to treat different forms of NCL as well as provide insight to novel therapeutic approaches in development for the NCLs

Searching for Novel Biomarkers Using a Mouse Model of CLN3-Batten Disease

CLN3-Batten disease is a rare, autosomal recessive disorder involving seizures, visual, motor and cognitive decline, and premature death. The Cln3Δex7/8 mouse model recapitulates several phenotypic characteristics of the most common 1.02kb disease-associated deletion. Identification of reproducible biomarker(s) to facilitate longitudinal monitoring of disease progression and provide readouts for therapeutic response has remained elusive. One factor that has complicated the identification of suitable biomarkers in this mouse model has been that variations in animal husbandry appear to significantly influence readouts. In the current study, we cross-compared a number of biological parameters in blood from Cln3Δex7/8 mice and control, non-disease mice on the same genetic background from multiple animal facilities in an attempt to better define a surrogate marker of CLN3-Batten disease. Interestingly, we found that significant differences between Batten and non-disease mice found at one site were generally not maintained across different facilities. Our results suggest that colony variation in the Cln3Δex7/8 mouse model of CLN3-Batten disease can influence potential biomarkers of the disease

The clinical relevance of PCL index on the reconstruction of anterior cruciate ligament with hamstring tendon autograft

The posterior cruciate ligament index (PCL index) has been reported as a diagnostic and prognostic marker for anterior cruciate ligament (ACL) reconstruction. The clinical relevance of PCL index on the reconstruction of ACL with hamstring tendon autograft has not been described in the literature. The objective of this study is to evaluate the importance of the PCL index as a marker of anatomic reconstruction and of functional improvement of patients undergoing ACL reconstruction with HT autograft. Twenty-four patients were submitted to ACL reconstruction with HT autograft. The PCL index was assessed by magnetic resonance imaging before and after surgery. The functional evaluation was performed through the International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form© and Knee Society Knee Scoring System© (IKS). Patients presented a significant positive variation of the PCL index, IKDC and IKS scores. There is no significant correlation between PCL index variation and IKDC and IKS scores (p > 0.05). Unlike other studies reporting a relationship between the PCL index, control of rotational kinematics, and functional improvement in patients undergoing ACL reconstruction with bone-patellar tendon-bone autograft, this study does not demonstrate this association. There is evidence in this study to show that the PCL index may be used as an anatomic reconstructive marker of ACL but not to predict the clinical outcome in this type of reconstruction.(undefined

A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies

<div><p>The Neuronal Ceroid Lipofuscinoses (NCLs), also known as Batten disease, result from mutations in over a dozen genes. Although, adults are susceptible, the NCLs are frequently classified as pediatric neurodegenerative diseases due to their greater pediatric prevalence. Initial clinical presentation usually consists of either seizures or retinopathy but develops to encompass both in conjunction with declining motor and cognitive function. The NCLs result in premature death due to the absence of curative therapies. Nevertheless, preclinical and clinical trials exist for various therapies. However, the genotypes of NCL animal models determine which therapeutic approaches can be assessed. Mutations of the <i>CLN2</i> gene encoding a soluble lysosomal enzyme, tripeptidyl peptidase 1 (TPP1), cause late infantile NCL/CLN2 disease. The genotype of the original mouse model of CLN2 disease, <i>Cln2</i>^<i>-/-</i>, excludes mutation guided therapies like antisense oligonucleotides and nonsense suppression. Therefore, the purpose of this study was to develop a model of CLN2 disease that allows for the assessment of all therapeutic approaches. Nonsense mutations in CLN2 disease are frequent, the most common being <i>CLN2</i>^<i>R208X</i>. Thus, we created a mouse model that carries a mutation equivalent to the human p.R208X mutation. Molecular assessment of <i>Cln2</i>^{<i>R207X/R207X</i>} tissues determined significant reduction in <i>Cln2</i> transcript abundance and TPP1 enzyme activity. This reduction leads to the development of neurological impairment (e.g. tremors) and neuropathology (e.g. astrocytosis). Collectively, these assessments indicate that the <i>Cln2</i>^{<i>R207X/R207X</i>} mouse is a valid CLN2 disease model which can be used for the preclinical evaluation of all therapeutic approaches including mutation guided therapies.</p></div

Altered motor skills in 3-month-old <i>Cln2</i>^{<i>R207X/R207X</i>} mice.

<p>A panel of behavioral tests including modified vertical pole (A and B), rotarod (C), and modified hanging wire (D) were used to assess motor skills in 1 and 3-month-old <i>Cln2</i>^<i>+/+</i> (n = 14) and <i>Cln2</i>^{<i>R207X/R207X</i>} (n = 20) mice. The modified hanging wire was only evaluated at 3 months of age. (A) Climb down time as assessed by the vertical pole showed no differences at 1 month of age between <i>Cln2</i>^<i>+/+</i> and <i>Cln2</i>^{<i>R207X/R207X</i>} but at 3 months of age <i>Cln2</i>^{<i>R207X/R207X</i>} mice trended towards an increased time. (B) Time to turn downward, also assessed by the vertical pole, showed no differences at 1 month of age between <i>Cln2</i>^<i>+/+</i> and <i>Cln2</i>^{<i>R207X/R207X</i>}. By 3 months of age however, <i>Cln2</i>^{<i>R207X/R207X</i>} mice demonstrated significant difficulties turning around. (C) <i>Cln2</i>^<i>+/+</i> and <i>Cln2</i>^{<i>R207X/R207X</i>} mice exhibited no differences in their latency to fall from an accelerating rotarod at both 1 and 3 months of age. (D) 3-month-old <i>Cln2</i>^{<i>R207X/R207X</i>} mice have significant difficulties hanging from a wire rack. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (***p <u><</u> 0.001).</p

Lysosomal accumulation of mitochondrial ATP synthase subunit c in <i>Cln2</i>^{<i>R207X/R207X</i>} mice.

<p>Subunit c accumulation was detected by immunofluorescent staining. (A) Images of superficial and deep cortical layers demonstrate diffuse and pronounced accumulation of mitochondrial ATP synthase subunit c in 3-month-old <i>Cln2</i>^{<i>R207X/R207X</i>} mice. (B) Images from 3-month-old <i>Cln2</i>^<i>+/+</i> (n = 4) and <i>Cln2</i>^{<i>R207X/R207X</i>} (n = 5) mice were blindly collected and analyzed for differences in cell number, total number of immunoreactive puncta, number of puncta per cell, and average punctum area. <i>Cln2</i>^{<i>R207X/R207X</i>} mice have significantly increased total number of puncta, puncta per cell, and punctum size. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (**p <u><</u> 0.01, ***p <u><</u> 0.001, and **** p < 0.0001).</p

Frequency of CLN2 disease-associated mutations.

<p>(A) Allele frequency of CLN2 disease mutations demonstrates a predominance of nonsense (29%) and missense (22%) mutations. (B) The most common CLN2 disease mutations consists of either the intronic transversion c.509-1G>C that results in altered transcript splicing or the exonic transition c.622C>T that results in the p.R208X nonsense mutation.</p

Decreased TPP1 activity in various <i>Cln2</i>^{<i>R207X/R207X</i>} mouse tissues.

<p>Fluorogenic TPP1 enzyme activity assays were used to measure endogenous levels of TPP1 activity from five different tissues obtained from 1-month-old <i>Cln2</i>^<i>+/+</i> (n = 3) and <i>Cln2</i>^{<i>R207X/R207X</i>} (n = 3) mice. Four technical replicates were performed using the three biological samples obtained from <i>Cln2</i>^<i>+/+</i> and <i>Cln2</i>^{<i>R207X/R207X</i>} mice. <i>Cln2</i>^{<i>R207X/R207X</i>} TPP1 activity was normalized to <i>Cln2</i>^<i>+/+</i> TPP1 activity. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (** p <u><</u> 0.01, ***p <u><</u> 0.001, and **** p < 0.0001).</p

Genetic design of the CLN2 disease mouse model, <i>Cln2</i>^{<i>R207X/R207X</i>}.

<p>(A) A <i>Cln2</i> targeting vector carrying the c.619C>T mutation (Line 2), also known as p.R207X mutation, was electroporated into C57BL/6x129S6/SvEv embryonic stem cells. Homologous recombination between wildtype <i>Cln2</i> (Line 1) and the targeting vector (Line 2) resulted in the generation of a <i>Cln2</i>^{<i>Neo;R207X</i>} allele (Line 3). Removal of the <i>Neo</i> cassette via FRT-flippase mediated excision (FRT sites represented by grey arrows flanking the <i>Neo</i> cassette) generated the <i>Cln2</i>^<i>R207X</i> allele (Line 4) resulting in <i>Cln2</i>^{<i>+/R207X</i>} mice. <i>Cln2</i>^{<i>+/R207X</i>} mice were bred to create <i>Cln2</i>^{<i>R207X/R207X</i>} mice. (B) <i>Cln2</i> sequencing results confirm the retention of the mutation following breeding.</p

Diffuse cortical astrocytosis in <i>Cln2</i>^{<i>R207X/R207X</i>} mice.

<p>(A) Images of superficial and deep cortical layers from 3-month-old <i>Cln2</i>^<i>+/+</i> and <i>Cln2</i>^{<i>R207X/R207X</i>} mice demonstrate increased GFAP immunostaining and enlarged astrocytes. (B) Images from 3-month-old <i>Cln2</i>^<i>+/+</i> (n = 4) and <i>Cln2</i>^{<i>R207X/R207X</i>} (n = 5) mice were blindly collected and analyzed for GFAP immunostaining intensity, GFAP staining area, and astrocyte area. <i>Cln2</i>^{<i>R207X/R207X</i>} mice have significantly increased GFAP immunostaining and astrocyte size relative to <i>Cln2</i>^<i>+/+</i>. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (*p <u><</u> 0.05, **p <u><</u> 0.01, ***p <u><</u> 0.001, and **** p < 0.0001).</p