Sex-split analysis of pathology and motor-behavioral outcomes in a mouse model of CLN8-Batten disease reveals an increased disease burden and trajectory in female Cln8^mnd mice

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited lysosomal diseases that result in neurodegenerative disease within pediatric and adult populations. Commonly known as Batten disease, NCLs have an extensive range of phenotypic presentation, although most forms can be clinically characterized by declining cognitive and motor functions, ocular dysfunction, and eventual blindness, epilepsy, and a decreased lifespan [1] (for a recent review see [2]). Although NCLs are considered rare in nature, together they are the most prevalent neurodegenerative disease in the pediatric population with an estimated incidence of 2–4/100,000 births [3, 4] and an even greater incidence within certain populations. The etiology of NCLs is due to a mutation in one of at least 13 currently identified ceroid lipofuscinosis neuronal (CLN) genes—often encoding enzymes or regulatory proteins involved in proper lysosomal function [5, 6]. One of these genes, CLN8, encodes a transmembrane endoplasmic reticulum (ER) protein (CLN8) that has been shown to be involved in the trafficking of lysosomal-destined enzymes between the ER and Golgi, in addition to integral involvement with other lysosomal processes such as biogenesis [6, 7]. Additionally, studies have demonstrated neuronal-specific roles of CLN8 in neurite maturation, differentiation, and support of various neuronal populations [7‐9]. Mutations in CLN8 results in characteristic NCL symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and other neurodegenerative signs [6, 10].

CLN8 Batten disease (CLN8 disease) is a variant late-infantile form of Batten disease with an onset of symptoms generally between 5 and 10 years old [11]. Patients with CLN8 disease present with progressive deterioration of motor and cognitive abilities, visual symptoms, and epileptic seizures [6]. Two classic variants arising from mutations of CLN8 have been well described: (1) “Northern Epilepsy” is a condition characterized by epileptic seizures (tonic–clonic and/or complex partial) with peak frequency in adolescence followed by declining cognition and deteriorating motor skills due to cerebellar atrophy [12, 13]. Hirvasniemi et al. first identified Northern Epilepsy within patients of Northern Finland where patients all shared a homozygous missense mutation of CLN8 [13], but this subtype has also been described to result from other mutations in other populations [14, 15]; (2) Variant Late-infantile NCL (vLINCL) is a more severe phenotype associated with CLN8 mutation first identified in Turkish families. This variant typically presents as epileptic seizures, motor and cognitive deterioration, and visual disturbances (which help distinguish it from Northern Epilepsy clinically). Furthermore, patients with vLINCL experience more severe disease progression with motor and cognitive deterioration occurring within several years, as compared to Northern Epilepsy which progresses over several decades [16]. Despite these two well-described phenotypes of CLN8 disease within distinct populations, cases have been described in a multitude of geographic locations throughout the world with variability in disease progression [14, 15, 17‐21]. As such, clinical presentation of CLN8 disease may not always fall into a discrete category and suspicion of the disorder warrants further genetic and diagnostic testing [16].

Recently, greater emphasis has been placed on understanding and identifying sex distinctions as an important modulator of physiology, anatomy, and pathology in disease, including within various forms of Batten disease [22‐25]. A multitude of neurodegenerative diseases demonstrate sex biases, such as greater prevalence of Alzheimer’s disease in women and increased prevalence of Parkinson’s disease and amyotrophic lateral sclerosis in men [26]. The field of Batten disease is no different: NCLs have been shown to demonstrate sex-based clinic and pathologic differences in patients and in animal models. Although male subjects typically experience earlier disease onset, females with juvenile NCL (JNCL; CLN3 Disease) suffer a more rapid disease progression characterized by quicker cognitive decline, loss of motor coordination, and earlier death [27, 28]. Further, Cialone et al. [28] described female patients as having a poorer quality of life due to greater physical impairment. Overall, identifying sex differences (or lack thereof) in humans with Batten disease is exceedingly difficult due to various mutations within the range of CLN genes and complex interactions between their respective unique genetics and environment.

The utilization of murine models in Batten disease research has greatly expanded the ability to investigate sex differences in this family of diseases, in addition to highlighting the importance of sex as a factor to be considered when designing and analyzing therapeutic trials [29]. For instance, sex-dependent differences in gene expression response to galactosylceramide were found in the Cln3^Δex7/8 murine model [30]. Further, Poppens et al. described female Cln6^nclf mice to experience accelerated disease progression, more severe behavioral issues and motor decline, and differences in histopathological effects [31]. A prior investigation of Cln8^mnd mice revealed sex differences in retinal vulnerability where female retinas exhibited higher oxidation rates and caspase-3 mediated apoptosis, in addition to a more severe histopathological profile of the retina [32]. However, the disease associated phenotypes in relationship to sex examined in this study were limited to visual deficits in the Cln8^mnd mouse model. To add to this body of work, we examined the influence of sex on psychomotor behavioral outcomes and histopathology within thalamus and primary somatosensory cortex of Cln8^mnd mice. Additionally, Cln8^mnd sexual dimorphisms in AAV9 gene therapy response were also explored.

This study demonstrates sex differences in the progression of CLN8 disease in the Cln8^mnd murine model. Specifically, female Cln8^mnd mice performed worse on the MWM assessment, perished earlier, and showed increased astrocyte and microglia reactivity over their Cln8^mnd male counterparts at several time points. Our reported results of ASM and SubC accumulation comparisons between Cln8^mnd male and female mice demonstrated contrasting data in that storage accumulation was more pronounced at different time stages of pathogenesis. Generally, Cln8^mnd male mice had greater ASM accumulation within the VPM/VPL and S1BF whereas Cln8^mnd female mice had greater SubC burden within both areas and the striatum. Accumulation is thought to occur due to any disruption in the basic processes of autophagy, lysosomal function, or oxidative damage; however, other mechanisms of accumulation may exist [38]. The primary storage material of ASM within CLN8-Batten disease is SubC, although, other disease subtypes may have a differing primary constituent like sphingolipid activator proteins in CLN1 and CLN10-Batten disease [38, 39]. Other ASM accumulation components include neutral lipids, phospholipids, dolichol pyrophosphate linked oligosaccharides, lipid linked oligosaccharides, dolichol esters, and metal ions [38, 40, 41]. Based on our data suggesting Cln8^mnd female mice having a greater SubC component of ASM, it is thus presumed their male comparisons are accumulating other molecular components from an unknown mechanism.

Interestingly, there was a marked increase in glial activity between 2 and 4 months of age, indicating this may be the critical time point in which pathological change from both these processes occurs. It is possible that this increase in gliosis may be a contributing factor to the poorer MWM performance and decreased lifespan seen within Cln8^mnd females. Previous investigations of neural injuries in mice offer support for an association between enhanced gliotic activity and poorer motor-behavioral outcomes in assessments like the MWM [42‐44]. However, it is worth noting that prior investigation of sex differences of a CLN6 disease mouse model revealed Cln6^nclf males experience greater microgliosis than Cln6^nclf females at 6 months of age within the S1BF despite Cln6^nclf females perishing earlier and exhibiting poorer motor-behavioral outcomes [31]. These differences in pathological variations, such as increases in male ASM versus female SubC and increases in female gliosis in one NCL model versus male gliosis in another, highlight the complexity of interpreting pathological changes and their relation to disease progression and treatment outcomes, and specifically suggest that a more holistic approach may be required for this purpose. Unfortunately, these sex-dependent murine model differences cannot be correlated with clinical outcomes in humans with CLN8 disease since there have been no such detailed human investigations, likely due to small patient populations and the difficulty in comparing human subjects due to environmental differences and genetic heterogeneity of CLN8 mutations [45].

Greater pathological visual deficits and/or dysfunction are another possible explanation for worse MWM performance by Cln8^mnd females. Cln8^mnd females previously demonstrated harsher retinal histopathologic profiles and retina cell apoptosis compared to Cln8^mnd male comparisons [32], and we hypothesize these differences and increased activated glia may contribute to Cln8^mnd females’ greater visual aberrations and poorer performance [46, 47]. Prior investigations have highlighted glial dysfunction in NCL murine models coinciding with subsequent neuronal damage of the visual cortex and retina, resulting in deterioration of visual perception and retinal function [48‐50]. Moreover, attenuation of inflammatory microglia via therapeutic agents in Batten disease animal models improved visual acuity, reduced retinal thinning, and improved retinal ganglion cell survival [49, 51‐53]. Sex comparisons of microglia contribution to pathology and response to therapy in vision related systems may better elucidate this process.

An increasing body of evidence indicates that aberrant glial cell function contributes to the disruption of CNS homeostasis and resulting neurodegeneration in Batten disease [54, 55]. Broadly, activation of astrocytes and microglia predicts subsequent neuron degeneration within the local area in various Batten disease models, and in the Cln8^mnd mouse model specifically, enhanced gliosis coincides with further disease progression [10, 56]. More recently, investigation of in vitro glial cultures derived from CLN1 and CLN3 murine models demonstrates the negative influence of glia on neuron survival through differing phenotypic functional states [57‐59]. Ppt1^−/− microglia cultures were shown to exist in a basally activated state with increased secretion of cytokines and chemokines that induce neuron death, and similarly, cultured Cln3^Δex7/8 microglia behave in a reactionary state where stimuli elicit a caspase-1 mediated pro-inflammatory response that includes cytokine/chemokine production, glutamate release, and hemichannel activity that induces cell death [57, 59]. Furthermore, depletion of microglia via pharmacologic targeting can improve CLN1 disease in mice, and interestingly, Berve et al. observed surprising sex and anatomical region biases: greater preservation of Ppt1^−/− female microglia was observed as they were less responsive to pharmacologic treatment, especially within the S1BF, and females experienced subsequently poorer treatment outcomes compared to their male counterparts [51].

Nonetheless, the question remains why Cln8^mnd females exhibit enhanced microglial activation within the S1BF and VPM/VPL nuclei of the thalamus. Within murine brains, sexual dimorphism has been noted in microglia function, morphology, and colonization of brain structures–stemming from variance in sex-specific gene expression, circulating sex steroidal hormones and response to hormones, and epigenetic interactions [60‐63]. Female-derived mouse microglia tend to be more reactive and inflammatory than male-derived microglia, characterized by higher inflammatory cytokines, inflammatory-related receptor expression, and differential expression of estrogen receptor subtypes [61]. Comparison of microglial number within the amygdala, hippocampus, and parietal cortex revealed that male mice had more microglia in the initial post-natal period, coinciding with their testosterone surge, until the transition into adolescence when females exhibited greater microglia with an activated phenotype in the same regions [60]. The sex differences in microglial colonization may be influenced by disparate levels of sex hormones and chemokines, as evidenced by a 200-fold influx of CCL20 and 50 fold increase of CCL4 during the testosterone surge in early male mouse development [60, 64, 65]. Therefore, sex-dependent chemokine expression in Cln8^mnd mice is a possible explanation for the relatively increased microgliosis observed in Cln8^mnd females at later life stages, and should be investigated further.

Sexual dimorphism in genetic architecture and X-chromosome gene regulation may promote the chronic inflammatory process in Batten disease, and thus may partially explain the exacerbated phenotype observed within females [66‐69]. The X-chromosome is the locus of numerous genes related to immune function and regulation and through mechanisms like mosaic X-chromosome inactivation and “gene escape” from the inactivated X-chromosome, may lead to differential and bi-allelic expression of proinflammatory genes respectively [67, 68, 70, 71]. An estimated 3–7% and 15–23% of genes on the inactivated X-chromosome escape in female mice and humans respectively [70, 72, 73]. For example, cluster of differentiation (CD) 40 and 99 ligand are expressed on the X-chromosome and increased CD receptor-CD ligand engagement activates proinflammatory cascades involving T and B cells, monocyte derivatives like macrophages and microglia, and cytokine upregulation which is implicated in a multitude of neurologic disease [74, 75]. To our knowledge, no such studies have investigated the degree to which X-chromosome inactivation escape may influence the poorer histopathologic and motor-behavioral outcomes observed within female sex in Batten disease. Elucidation of the likely mechanism(s) by which this process occurs may provide insight for potential therapeutic targets to alleviate disease burden.

We also reported that AAV9 gene therapy was well received and generally efficacious to the same degree in Cln8^mnd mice regardless of sex, with one exception where AAV9-treated female mice performed worse on MWM assessments than their male counterparts, which as discussed may be due to the relatively worse retinal damage experienced by Cln8^mnd females [32]. There have been few publications on sexual dimorphism in AAV-mediated gene therapy, though reports have indicated differences in tissue transduction depending on serotype, route of administration, tissue type, and the presence of single or double-stranded genomes, with the most commonly affected tissues being the liver, skeletal muscle, and gonads [76‐78]. Specifically, one detailed report described how male-specific increases in liver transduction were the result of androgen-dependent pathways, and that modulating these pathways led to improved transduction in the livers of female mice [79]. While there is limited data on sex-dependent differences of AAV-gene therapy in the CNS, one recent study demonstrated sex-specific responses to intracerebroventricularly delivered AAV9 in a mouse model of Dravet syndrome, a debilitating seizure disorder caused by mutations in the α subunit of NaV1.1 channels (SCN1A) [80]. The authors speculated that these sex-specific differences occurred due to basal differences in voltage-gated sodium channel presence in male and female mice, indicating that any sex-specific differences in response to gene therapies, or lack of differences, may be due to whether there are sexually dimorphic functions already present for the protein product in question.

Taken together, the results from this investigation provide further evidence of sex-dependent differences in lifespan, histopathology, and motor-behavioral outcomes within the Cln8^mnd mouse model of Batten disease, and gives insight into sex-dependent responses to CNS-delivered AAV9 gene therapy. Although sex discrepancies have been observed in human subjects with CLN3-Batten disease, sparse information exists for other forms of NCLs. As such, based on the surmounting body of evidence demonstrating the importance of sex as a biologic modifier, prospective and retrospective analysis of sex differences in other forms of Batten disease should be conducted to yield a better understanding of disease pathogenesis and treatment response.

The majority of the data presented in this manuscript was previously published as a mixed-sex cohort in Johnson et al. [6] where the authors showed AAV9-gene therapy of CLN8 prevented CLN8 Batten disease characteristics within Cln8^mnd mice. The present manuscript primarily examines that previously collected data as a sex-split cohort, and adds additional analyses not previously published.