1. Stine N Hansen 1,2, Noreen A Alexander 1,2, Carina von Schantz 3, Outi Kopra 3, Anu Jalanko 3, Jonathan D Cooper 1,2 Neuropathological and reactive changes in aged Cln5 deficient mice 1 Pediatric Storage Disorders Laboratory, and 2 Department of Neuroscience, Institute of Psychiatry, King's College London, UK; 3 National Public Health Institute, Department of Medical Genetics and Molecular Medicine, Biomedicum Helsinki, Finland. REGIONAL VOLUMECORTICAL THICKNESSLAMINAR THICKNESS NEURONAL NUMBER ASTROCYTOSIS MICROGLIAL ACTIVATION Counts of neuronal number revealed no significant loss of neurons in laminae II & III, lamina IV or lamina V of somatosensory barrelfield cortex (Figure 4A). However, compared to control mice there was a profound loss of VPM/VPL thalamic neurons (Figure 4B) in 12 month old Cln5-/- mice. To survey effects on glial cell populations in 12 month old Cln5-/- mice we used immunohistochemical staining for markers of astrocytosis (GFAP) and microglial activation (F4/80). These markers revealed widespread astrocytosis (Figure 5) and a more localized microglial activation (Figure 6) within individual thalamic nuclei and other subcortical structures, together with the cortex. Quantitative thresholding image analysis confirmed these pronounced effects on both astrocyte and microglial cell populations in the interconnected thalamic relay nucleus VPM/VPL and primary somatosensory cortex (S1BF). Conventional epifluorescence illumination revealed widespread intracellular accumulation of autofluorescent storage material within neuronal soma throughout the CNS of aged Cln5 deficient mice (Figure 7). The neuronal ceroid lipofuscinoses (NCLs) are a significant cause of progressive intellectual and neurological deterioration during childhood. Collectively, this group of up to nine genetically distinct disorders (CLN1-CLN9) is considered the most common pediatric neurodegenerative encephalopathy. Clinically, the NCLs are typified by their progressive nature, presenting with visual failure leading to blindness, neurocognitive impairment, severe seizures and premature death. Cases usually present during childhood and are distinguished as infantile (INCL), late infantile (LINCL) and juvenile (JNCL). However, rare adult forms and several variant forms are also recognized. The pathological hallmark of the NCLs is the intralysosomal accumulation of autofluorescent storage material. Finnish variant LINCL (vLINCL Fin ) is the result of mutations in the Cln5 gene. Cln5 null mutant mice (Cln5-/-) are a useful model for studying the underlying pathological mechanisms in this disorder. To gain a detailed series of pathological landmarks from severely affected Cln5-/- mice, we have undertaken a stereological analysis of neurodegenerative and reactive changes in the CNS of 12 month old mutant mice. INTRODUCTION Aged Cln5-/- mice displayed atrophy of the cortical mantle and hippocampus (Figure 1). This atrophy also extended to subcortical structures including the thalamus and striatum. Cortical thickness measurements revealed a widespread and significant atrophy of the cortical mantle (Figure 2). This thinning of the cortex differed in extent between regions, with effects that extended to individual laminae (Figure 3). To explore the basis for these laminar- specific events we obtained optical fractionator estimates of neuronal number in the primary somatosensory barrelfield (S1BF) and the ventral posterior thalamic nucleus (VPM/VPL) which sends projections to S1BF (Figure 4). RESULTS We have examined the CNS of 12 month old Cln5-/- mice and age-matched control mice (+/+) on the same mixed strain background. To determine the extent of neuropathological changes at both regional and cellular levels we examined the expression of different immunohistochemical markers of neuronal and glial phenotype, using unbiased stereological methods and thresholding image analysis. Figure 1 Significantly reduced volume of the cortex, hippocampus, striatum and thalamus in 12 month old Cln5 -/- mice vs. age-matched controls (+/+) is revealed by Cavalieri estimates. * p<0.05 one-way ANOVA Figure 2 Widespread shrinkage of the cortical mantle in 12 month old Cln5-/- mice vs. age-matched controls (+/+). Cortical subfields surveyed include primary somatosensory barrelfield (S1BF), primary motor (M1), primary visual (V1) and lateral entorhinal (Lent) cortex. *** p<0.001, one-way ANOVA. Figure 3 Laminar-specific changes in cortical thickness in 12 month old Cln5-/- mice compared with age-matched controls (+/+, A-D). Laminar thickness measurements in primary motor cortex (M1, A), primary somatosensory barrelfield cortex (S1BF, B), primary visual cortex (V1, C), lateral entorhinal cortex (Lent, D). * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA. 12 month old Cln5 deficient mice exhibit a severe neurodegenerative and reactive phenotype that is more pronounced compared to results from a study in younger Cln5-/- mice. These data emphasize the progressive nature of the NCLs. Consistent with a mouse model of JNCL (Cln3 null mutant), Cln5-/- mice display a profound loss of sensory relay thalamic neurons, yet no loss of their target neurons in lamina IV of somatosensory cortex. Our preliminary data suggest that this vulnerability of thalamic neurons is an early event in pathogenesis. Cln5 deficient mice also exhibit pronounced glial responses within individual thalamic nuclei, which appear to occur very early in disease progression. It will be important to determine the relationship between these glial responses and neuronal loss, and whether this relationship differs between mouse models. These data provide further evidence for neurodegenerative and reactive changes in the thalamocortical system in mouse models of NCL and emphasize the localized nature of these events. CONCLUSIONS Figure 4 (A) Unbiased optical fractionator estimates of neuronal number in individual laminae of primary somatosensory barrelfield cortex (S1BF, A) and the ventral posterior thalamic nucleus (VPM/VPL, B) in 12 month old Cln5- /- mice and age-matched controls (+/+). Note the significant loss of thalamic relay neurons with no loss of their cortical target neurons. Figure 5 Thresholding image analysis confirms the pronounced up regulation of the astrocyte marker GFAP in the cortex and thalamus of 12 month old Cln5-/- mice vs. control littermates (+/+). Areas surveyed include somatosensory barrelfield cortex (S1BF), and the ventral posterior thalamic nucleus (VPM/VPL). *** p<0.001, one-way ANOVA. Figure 6 Thresholding image analysis confirms the pronounced up-regulation of the microglial marker F4/80 in the cortex and thalamus of 12 month old Cln5-/- mice vs. control littermates (+/+). Areas surveyed include somatosensory barrelfield cortex (S1BF), and the ventral posterior thalamic nucleus (VPM/VPL). *** p<0.001, one way ANOVA. METHODS AKNOWLEDGEMENTS This work was supported by European Commission 6 th Framework Research Grant LSHM- CT-2003-503051 (JDC), National Institutes of Health (NINDS) grant NS41930 (JDC), and grants to JDC from The Natalie Fund, Batten Disease Support and Research Association, Batten Disease Family Association, Batten Disease Research Trust and the Remy Fund. STORAGE MATERIAL Figure 7 Widespread intracellular accumulation of storage material in 12 month old Cln5-/- mice vs. littermate controls (+/+). The storage material of Cln5 deficient mice fluoresces at both rhodamine (Rho) and FITC filter sets and appears yellow in merged images. Representative images are coronal sections through the primary somatosensory barrelfield cortex (A-D) and sagittal sections through the cerebellum (E-H).