The specific loss of GnRH-positive neurons from the hypothalamus of sheep with CLN6 neuronal ceroid lipofuscinosis occurs without glial activation and has only minor effects on reproduction

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Abstract

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are characterized by progressive neurodegeneration resulting in widespread brain atrophy. Each form is assumed to be the consequence of some universal intracellular event; however, time course studies on the cerebral cortex of a sheep model of the CLN6 form revealed distinct regional neurodegeneration preceded by regional glial activation, spreading from quite localized foci. Previous neurological investigations have concentrated on obviously affected cortical functions. This study investigated the impact of ovine CLN6 NCL on a subcortical structure and function, the discrete gonadotrophin-releasing hormone (GnRH) secreting neurons of the hypothalamus, and the effect of changes in the neuroendocrine system on reproductive efficiency and embryonic development. The number of immunopositive GnRH neurons in the hypothalamus and median eminence of affected sheep was reduced by 80%, but the rest of the hypothalamus showed no changes or atrophy. This specific loss of neuron type was not accompanied by either microglial or astrocyte activation, which was absent from the hypothalamus and was not associated with cell-type-specific storage body accumulation. Ovarian responsiveness to follicle stimulating hormone, ovulation rates, sperm production, fertilization rates, embryonic development, and reproductive efficiency were sub-par but reproduction was still functional. This remains when the sheep are profoundly blind. We conclude that physiological functionality and connectivity, not genotype, determine neuron fate in CLN6 NCL.

Research Highlights

► The GnRH neuron population in the sheep model of CLN6 is reduced by 80%. ► The loss of GnRH neurons is not accompanied by glial activation. ► Reproduction performance of affected sheep is subnormal.

Introduction

Batten disease (the neuronal ceroid lipofuscinoses, NCLs) is a group of fatal inherited neurodegenerative diseases in children. Clinical symptoms include loss of sight, seizures, and mental deterioration with ages of onset ranging from congenital to adult (Haltia, 2003, Haltia, 2006, Goebel et al., 1999). Mutations in at least eight different genes underlie the different forms (www.ucl.ac.uk/ncl). Defining characteristics are profound neurodegeneration associated with severe brain atrophy and the accumulation of specific proteins in lysosomally derived storage bodies in most cells. In most forms, the accumulating protein is subunit c of mitochondrial ATP synthase, while the sphingolipid activator proteins, SAPs A and D, accumulate in the CLN1 infantile form (Chen et al., 2004, Goebel et al., 1999, Haltia, 2003, Haltia, 2006). Four genes, CLN1, CLN2, CLN5, and CTSD (CLN10), code for soluble lysosomal proteins (Sleat et al., 1997, Siintola et al., 2006, Tyynelä et al., 2000, Vesa et al., 1995), while the others, CLN3, 6, 7, and 8, encode membrane associated proteins (Ezaki et al., 2003, Heine et al., 2004, Lonka et al., 2004, Mole et al., 2004, Siintola et al., 2007). While the functions of these proteins are not yet defined, their locations and the commonality of subunit c storage (Palmer et al., 1997) support roles in a common ER–Golgi–endosome–lysosome pathway. There is also evidence for additional protein functions affecting synapses, the trafficking of synaptic vesicles, and synaptic pathology in some forms (Kyttälä et al., 2004, Lehtovirta et al., 2001, Luiro et al., 2001).

NCLs also occur in animals. Each form is represented by a murine analog (Cooper et al., 2006), and most studied of all animal forms is the naturally occurring CLN6 form in New Zealand South Hampshire sheep, caused by low CLN6 mRNA concentrations (Tammen et al., 2006).

While it has been assumed that the neurodegeneration is the consequence of some universal intracellular event, studies of the time course and regionality of neurodegeneration revealed that this is not the case. In the CLN6 affected sheep brain atrophy is most noticeable as degeneration of the cerebral hemispheres and the visual cortex is affected before, and more severely, than other cortical regions (Oswald et al., 2005). A close association was found between glial activation and subsequent neurodegeneration (Kay et al., 2006, Oswald et al., 2005). Glial activation has also been noted in murine models (Cooper et al., 2006, Kielar et al., 2007, Mitchison et al., 2004) and in other lysosomal storage diseases. Interneuron changes in the affected sheep follow the pattern of glial activation, and location and connectivity, not phenotype, determine GABAergic interneuron survival (Oswald et al., 2008). Storage body accumulation does not correlate with either neurodegeneration or glial activation.

The extent of atrophy of the cerebellum varies between disease forms and species but is generally less and begins later than cerebral atrophy. The affect of the disease on the thalamus is less conspicuous. Microglial activation and storage body accumulation have been noted in the lateral geniculate nuclei in CLN6 sheep (Oswald et al., 2005) and in three other thalamic nuclei in CLN1 mice (Kielar et al., 2007). There are functional linkages between the affected thalamic nuclei and the associated cortical regions. For instance, the lateral geniculate nuclei process visual input signals prior to visual cortex processing.

The hypothalamus is involved in homeostatic regulation and incorporates some small defined nuclei and less defined regions including the periventricular zone of the third ventricle and the medium eminence. It plays a major role in the secretion of hormones into the portal circulation of the pituitary gland, which in turn regulate the gonadal, mammary gland, thyroid, and adrenal cortex functions.

Anecdotal evidence of reproductive disturbances in NCL patients is supported by some clinical evidence that the hypothalamus or reproductive processes may be disturbed. Hypothalamic neurons were preserved in two deceased CLN2 patients but the soma tended to be hypertrophied (Kurata et al., 1999). Precocious reproductive development was reported in a young female CLN2 patient (Aysun et al., 2000) and increased hyperandrogenism and polycystic ovary syndrome together with an advancement of puberty in CLN3 female patients (Åberg et al., 2002).

Apart from breeding seasonality, the reproduction biology of humans and sheep is very similar and the CLN6 sheep model lends itself to a study of the hypothalamic–pituitary–gonad axis. Affected animals reach sexual maturity prior to the disease becoming terminal and are not treated with drugs to modify the neurological course of the disease that may cause other hormonal changes. The goals of this study were to investigate the gonadotrophin releasing hormone-positive (GnRH) neurons of the hypothalamus and the histology and functionality of the pituitary gland and gonads of sheep affected with CLN6 NCL, their embryonic development, and reproductive performance.

Section snippets

Animals

Sheep were maintained under standard New Zealand pasture conditions on a Lincoln University research farm. All animal procedures were carried out according to NIH guidelines and the New Zealand Animal Welfare Act, 1999. CLN6 affected South Hampshire lambs were generated by mating heterozygous ewes to homozygous affected rams and the genotype determined using a discriminatory c.822 G > A polymorphism in the CLN6 gene (Tammen et al., 2006). Both homozygous normal and heterozygous animals were used

Regional pathology in the hypothalamus and thalamus of adult affected sheep

A specific and dramatic loss of GnRH-positive staining from the periventricular region (Fig. 1) and medium eminence (Fig. 2) of the hypothalamus contrasted with the lack of other disease associated changes in this region. Staining in the periventricular region occurred primarily as either dots or beaded neurites, which were few enough to be counted easily and were morphologically similar in affected and control brains (Fig. 1). GnRH-positive cell bodies were seldom seen. Counting revealed an

Discussion

The specific loss of 80% of GnRH-positive staining from the hypothalamus of sheep affected with CLN6 NCL was remarkable from a number of points of view, not the least being the specificity of this loss. The hypothalamus is not a well-defined structure and GnRH neurons are scattered throughout the ventral region of the thalamus from the pre-optic area, level with the optic chiasma, through to the medium eminence. In addition, these neurons are found scattered near the wall of the third

Acknowledgments

We wish to thank Professor Alan Herbison, University of Otago, for the GnRH antiserum used in preliminary studies; Genevieve McQuire, Charles Hu, and Dennes Herrick for their help with the animal handling; Martin Wellby for running the hormone assays; and Dr. Laura Åberg, University of Helsinki, for encouraging this project. This work was supported by the United States National Institutes of Health grant no. NS053559.

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