Tracking sex-dependent differences in a mouse model of CLN6-Batten disease

Batten disease (neuronal ceroid lipofuscinoses) comprises a family of autosomal-recessive neurodegenerative diseases characterized by lysosomal accumulation of autofluorescent lipopigment [1‐4]. Pathological hallmarks of Batten disease include neuronal death in cortical and thalamic regions of the brain and massive gliosis throughout the CNS, presenting functionally as degeneration of vision, psychomotor delay, and premature death [5‐7]. CLN6-Batten disease, resulting from mutations in CLN6, constitutes two distinct diseases: a pediatric form, also referred to as variant late infantile neuronal ceroid lipofuscinoses, and a rare, less-severe adult-onset form referred to as Kufs type A disease [8, 9]. The pediatric variant of CLN6 disease begins between the ages of 18 months and 8 years, presenting with language impairment, motor deterioration and cognitive deficiencies, followed by vision loss, seizures, and ultimately premature death during the second decade of life [10]. There are a number of naturally occurring CLN6 animal models used in therapeutic development, including the Cln6^nclf mouse model that contains a similar point mutation as found in human patients and develops the classical pathophysiological hallmarks of Batten disease, such as intracellular inclusion, retinal degeneration, hind-limb paralysis, and premature death [11‐13]. Many of the past Batten disease studies have excluded female mice from therapeutic studies to avoid confounding variables related to sex hormones and chromosomal differences [14, 15]. However, these biological disease modifiers can potentially limit the translatability of mouse findings to female patients, as sex-based differences that may affect disease susceptibility, disease severity, and therapeutic efficacy [14]. For example, sex specific symptomatic differences have been reported in other neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and autism spectrum disorders [14, 16‐19]. In addition, patient response to therapeutics has varied by sex, as estrogen, testosterone, and other sex-linked genes may affect drug effectiveness [20].

In the Batten disease field, studies on CLN3-Batten disease, a genetically distinct subtype of Batten disease, have shown differences in disease progression in patients depending on sex [8, 9, 21, 22]. On average, female CLN3-Batten disease patients present with symptoms 1 year later than their male counterparts, have accelerated disease progression following symptom onset, and die 1 year earlier than males [21]. Initial characterization of the Cln3^Δ7/8 murine model did not consider sex-based differences, however, recent work with this model has demonstrated that females exhibit poorer performance in behavioral tests [23, 24]. Additionally, the naturally occurring mouse model of CLN8-Batten disease has shown sex differences in female Cln8^mnd mice, where female retinas exhibited higher levels of retinal oxidative stress and caspase-3 activity compared to males [25]. These findings prompted us to investigate sex discrepancies in outcomes associated with CLN6 disease, exploring differences in disease onset and progression between male and female Cln6^nclf mice. We describe subtle histopathological differences between the sexes and a more rapid disease progression in female Cln6^nclf mice. Consequently, including sex as a factor during studies and subsequent analyses can ensure proper development of therapeutic treatments for patients with CLN6 disease.

In this study, we show differences in the progression CLN6 disease between sexes in a Cln6^nclf mouse model. Female Cln6^nclf mice presented with an earlier increase subunit C accumulation, and subsequently performed more poorly on behavioral tests and perished earlier than their male counterparts. Male Cln6^nclf mice, on the other hand, showed an increase in ASM at an earlier time point, as well as an increase in microglial activity at 6 months of age. While there have been previous comprehensive natural history studies of CLN6 disease patients, interpretation of sex-based outcomes is limited due to varying CLN6 mutations [9, 26, 27]. In the CLN3 variant of Batten disease, where the delta 7/8 mutation is common and affects ~ 75% of patients, male patients display symptoms before female patients, though females ultimately progress more quickly and die prior to males [21, 28]. It’s possible that the momentary increase in ASM seen in male mice reflects an early disease presentation, though this doesn’t translate into earlier functional difficulties. As the molecular underpinnings of CLN6 disease are not well understood, the extent to which these pathological changes translate to behavior changes in Cln6 mice will need to be the subject of future study.

The observed sex driven differences in disease progression are not unique to the Cln6^nclf mice or Batten disease. While the biological basis for variance in disease progression between sexes is unknown, hormonal factors may contribute to the observed differences. Among adults with neurodegenerative diseases, estradiol appears to play a protective role in females [29, 30]. However, in adolescent females with juvenile Batten disease, estrogen may be doing the exact opposite: CLN3-Batten disease females of post-pubertal age, when estrogen levels are elevated, demonstrated earlier loss of independence, and thus, estrogen may be contributing to the rapid disease progression [21].

Batten disease is an immune-mediated disease characterized by chronic neuroinflammation that is sustained by persistent glial activation in the brain, leading to damage and death of neighboring neurons and glial cells. Gonadal hormones support coordination of neuron-glia interactions and regulate reactive gliosis and neuroinflammation [31‐33]. Control of reactive gliosis by progesterone and estradiol is well documented, yet gliosis regulation by androgens has not been extensively explored. In general, estradiol appears to reduce astrocyte activity in the cerebral cortex, though, this contradicts the female Cln6 mouse presentation of heightened astrocytosis. However, evidence suggests that testosterone decreases reactive astroglia and microglia after neuronal damage [31, 32, 34]. Further, female microglia have been shown to exhibit higher phagocytic capacity than males and proinflammatory conditions, while male microglia have more efficient migratory response [35, 36]. Because glial cells become cytotoxic when chronically activated, female reduction in microglial activity may be a sign of advanced disease progression as these glial cells may have become inactive [37]. Furthermore, sex differences in microglial number may result from differences in chemotactic signaling and consequent microglial recruitment in males and females. Indeed, males exhibit higher levels of chemokines CCL20 and CCL4 in the hippocampus and cortex during critical periods of development, while females have elevated levels of proinflammatory cytokine interleukin (IL)-1β [38]. However, chemokines have not been studied in great detail in a healthy brain or in Batten disease, and the extent to which they may play a part in neurodegeneration remains unclear. Additionally, females are more vulnerable than males to develop Alzheimer’s disease, and although androgens have been studied less extensively than estrogens, androgens exert anti-inflammatory effects on microglia in AD models [39, 40].

Sex-based differences in Batten disease may also be related to the rise of autoantibodies in females. Estrogen has been shown to increase autoantibodies in systemic lupus erythematosus, accelerating disease progression [41]. In Batten disease, a similar autoimmune response in the CNS of both animal and patient populations contributes to disease progression [21]. Overall, hormonal differences in males and females likely explain some sex-specific immune responses, modifying disease course. As suppression of the immune system has been used in preclinical and clinical Batten disease studies, the extent to which these therapies are beneficial in both sexes should be a point of focus in the future [42, 43].

Here, we provide the first sex comparison of pathological and behavioral differences in Cln6^nclf mice, finding notable differences between the sexes. Moreover, our findings are identical to observed by another laboratory working with the same CLN6 mutant strain (personal communication, Drs. Stephanie Hughes and Hannah Best). The Cln6^nclf mouse model echoes the accelerated disease progression reported of females with Batten disease, and this information will be instrumental in providing appropriate treatments to female Batten disease patients in the future [21]. Currently, no definitive treatments or cures exist for CLN6 disease, a rapidly-progressing neurodevelopmental disease. However, as potential therapeutics are being investigated, sex-related differences must be taken in to account to target translatability to women.