2.1.2.2 MODIFICACIONES FISIOLÓGICAS DE LA SEROTONINA

No overt neuropathology was detected in the ovine NCL cerebellum during the study. The gross morphology of the cerebella in both ovine disease models remained unaltered during the disease and the size of the affected cerebella approximated normal (Figure 4.3). End-stage MRI studies on the affected sheep corroborated this structural finding (Amorim et al., 2015; Sawiak et al., 2015). This contrasts with the characteristic cerebellar pathology reported in human CLN5 and CLN6. Cerebella from human CLN6 patients may be normal or show some atrophy (Peña et al., 2001; Cannelli et al., 2009), but it has been reported that the cerebellum in human CLN5 cases is severely atrophied, with an almost complete depletion of cerebellar granule and Purkinje cells observed post mortem (Tyynelä et al., 1997; Goebel et al., 1999; Haltia, 2003). Brain imaging of human CLN5 patients reveals atrophy of the cerebellum to be the most striking abnormality (Autti et al., 1992), yet only dilated cerebellar sulci without gross degeneration is seen in MRI or CT studies of a canine Border collie CLN5 model (Koie et al., 2004; Mizukami et al., 2012). Only mild cerebellar atrophy has been reported for Devon cattle with a naturally occurring CLN5 NCL (Jolly et al., 1992). The reason why cerebellar pathology does not manifest in ovine NCL, particularly CLN5, is unclear. The sheep in the current study did not die from the disease, being euthanised for humane reasons. It may be that the degenerative effects of the disease that manifest initially in the cerebral cortex of the ovine NCLs were simply delayed in the cerebellum and sheep did not live long enough to exhibit cerebellar pathology.

The effect of the disease on the thalamus is more contentious. Whilst the main pathological target in multiple forms of NCL is the cortex (Haltia, 2003; Palmer et al., 2013), there is evidence that the thalamus is affected earlier in the disease progression than the cortex in murine models of CLN1, 2, 3, 6, 8 and 10 (Pontikis et al., 2004, 2005; Weimer et al., 2006; Kielar et al., 2007; Partanen et al., 2008; Kuronen et al., 2012). In contrast though, neuronal loss in the cortex preceded that in the thalamus in the CLN5 knock-out mouse (von Schantz et al., 2009). MRI studies on humans show decreased T2 intensity in the thalamus at the time of diagnosis in a Costa Rican CLN6 patient (Peña et

al., 2001) and in some CLN5 cases (Holmberg et al., 2000) whilst an almost complete loss of thalamic

neurons has been reported in post mortem human CLN5 brain tissue at end-stage disease (Tynnelä et

al., 1997).

The thalamus represents a structure that harbours high levels of neural interconnectivity. Every sensory CNS system (with the exception of olfactory) includes a thalamic nucleus that receives sensory signals and relays them through thalamocortical projections to the associated primary cortical region. Reciprocal corticothalamic fibres then relay the processed feedback information to the respective thalamic nuclei, in a process termed ‘top-down’ processing (Rauschecker, 1998; Granger & Hearn, 2007).For instance, in the visual system, sensory input from the retina is sent to

80 the lateral geniculate nucleus (LGN) of the thalamus, which in turn projects to the visual cortex of the occipital lobe. In turn, the LGN receives strong feedback connections from the primary visual cortex (Cudeiro & Sillito, 2006; Sillito et al., 2006).

The current study is consistent with others of ovine NCL that showed that subcortical (including thalamic) degeneration was limited to very late in the disease progression at most (Mayhew et al., 1985; Oswald et al., 2005, 2008; Kay et al., 2011). Whilst sparse activated microglia and astrocytes were first evident from 12 months of age in the affected sheep thalami, this was significantly delayed compared with the cortical reactive changes which originated in the visual and parieto-occipital cortex. Only at late stage disease were GFAP and GSB4 expression levels upregulated in the reciprocal LGN (Figure 4.8and Figure 4.11). Nissl staining in the current study revealed no obvious qualitative changes in the subcortical or thalamic architecture and although MRI scans of CLN5 and CLN6 sheep reported some subcortical atrophy (Amorim et al., 2015; Sawiak et al., 2015), these imaging studies were performed at end-stage disease, when the entire brain was atrophied. Kay et

al. (2011) found the cross-sectional area of the CLN6 affected thalamus was reduced by only about

5%. The gross normality of the thalamus in the ovine NCLs compared with that in murine models would suggest that the pathogenic pathways may be species-specific (Kay et al., 2011) and any disruption in the thalamocortical network in affected sheep would result from the cortical degeneration rather than vice versa.

Interestingly, the cortical laminae targeted in CLN5 and CLN6 NCL seem to differ between species. Layer V neuron vulnerability has been reported for human and murine CLN6 (Elleder et al., 1997) whilst the laminar loss of neurons in human CLN5 is targeted to laminae III and V (Tyynelä et al., 1997) and to laminae IV-VI in CLN5 knock-out mice (von Schantz et al., 2009). As neurons within the individual cortical laminae are morphologically unique, with specific axonal projections to different CNS regions, one might expect neuronal cell loss within each lamina to produce distinct phenotypes. For instance, commissural and associative neurons reside within the laminae II and III which are most affected in the ovine NCLs. These neurons have projections across the midline to the contralateral hemisphere or to other ipsilateral cortical locations respectively (Greig et al., 2013). Potentially their loss could have significant consequences on inter-hemispheric and intracortical co-ordination of neuronal activity. The deeper neuronal laminae V and VI contain pyramidal and multiform neurons which mainly send efferent projections to subcortical structures, such as the basal ganglia and thalamus. Loss of these neurons would likely disrupt the reciprocal interconnections between the cortex and subcortex.