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Astrocytosis in infantile neuronal ceroid lipofuscinosis: friend or foe? Charles Shyng* and Mark S. Sands*†1 *Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A. †Department of Genetics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
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Abstract Infantile neuronal ceroid lipofuscinosis (INCL; infantile Batten disease) is an inherited paediatric neurodegenerative disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1) and is thus classified as a lysosomal storage disease. Pathological examination of both human and murine INCL brains reveals progressive, widespread neuroinflammation. In fact, astrocyte activation appears to be the first histological sign of disease. However, the role of astrocytosis in INCL was poorly understood. The hallmark of astrocyte activation is the up-regulation of intermediate filaments, such as glial fibrillary acidic protein (GFAP) and vimentin. The role of astrocytosis in INCL was studied in a murine model lacking PPT1 and the intermediate filaments GFAP and vimentin (triple-knockout). This murine model of INCL with attenuated astrocytosis had an exacerbated pathological and clinical phenotype. The triple-knockout mouse had a significantly shortened lifespan, and accelerated cellular and humoural neuroinflammatory response compared with the parental PPT1 − / − mouse. The data obtained from the triple-knockout mouse strongly suggest that astrocyte activation plays a beneficial role in early INCL disease progression. A more thorough understanding of the glial responses to lysosomal enzyme deficiencies and the accumulation of undergraded substrates will be crucial to developing effective therapeutics.
Lysosomal storage diseases Lysosomal storage disorders (LSDs) are a group of inherited metabolic diseases typically caused by a malfunction in the lysosome. Most LSDs are inherited in an autosomal recessive fashion and are due to deficiencies in lysosomal enzymes. Deficiencies in lysosomal enzyme activity result in the accumulation of non-degraded or partially degraded macromolecules. Although most storage accumulation is a direct result of aberrant or complete lack of enzyme activity, mutations to genes coding transport proteins and posttranslational modifiers can also result in an LSD phenotype [1]. The progressive accumulation of storage material is associated with a broad spectrum of cellular and clinical manifestations, such as organomegaly, neurodegeneration, skeletal dysplasia, lipid accumulation and developmental defects. Although each LSD has its own unique set of clinical symptoms, approximately two-thirds of LSDs have a neurological component [2].
Neuronal ceroid lipofuscinoses The neuronal ceroid lipofuscinoses (NCLs), or Batten disease, are a subset of LSDs caused by mutations to at least 13 different genes. Classified as the most common Key words: astrocytosis, glial fibrillary acidic protein, infantile neuronal ceroid lipofuscinosis, intermediate filament, lysosomal storage disease, vimentin. Abbreviations: CNS, central nervous system; GFAP, glial fibrillary acidic protein; INCL, infantile neuronal ceroid lipofuscinosis; LSD, lysosomal storage disorder; NCL, neuronal ceroid lipofuscinosis; PPT1, palmitoyl-protein thioesterase-1. 1 To whom correspondence should be addressed (email [email protected]).
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inherited paediatric neurological disorder (∼1 in 12 500 live births), NCL patients present with visual deficits, loss of motor co-ordination, severe cognitive decline and seizures [3]. Pathologically, the NCLs are defined by the accumulation of autofluorescent material in many cells of the body, but may be most pronounced in the brain [4]. Though the NCLs share clinical and histological presentations, particularly the progressive deterioration of the central nervous system (CNS), individual NCLs are distinct and are differentiated by genetic mutation, age, severity and ultrastructural characterization of storage material [5].
Infantile NCL Infantile neuronal ceroid lipofuscinosis (INCL; infantile Batten disease) is the earliest onset and most aggressive form of the NCLs. INCL is caused by an array of mutations in the CLN1 gene that lead to a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase1 (PPT1) [6]. The normal role of PPT1 is to cleave palmitoyl fatty-acyl chains from modified cysteine residues. Deficiency in PPT1 leads to the accumulation of the hallmark autofluorescent storage material, characterized ultrastructurally as granular osmiophilic deposits (GRODs). This accumulation is prevalent in all cell types, though it appears to predominate in the CNS [7]. The clinical presentation of INCL is similar to that of other NCLs, with the first clinical sign being blindness, followed by cognitive decline, motor dysfunction and intractable seizures. Initially, Biochem. Soc. Trans. (2014) 42, 1282–1285; doi:10.1042/BST20140188
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the patients develop normally and symptoms present between 6 and 12 months of age. By 3–4 years of age, patients typically enter a vegetative state and death occurs between 4 and 6 years of age. Autopsy reveals that INCL children have severe cortical atrophy with the brain mass equal to ∼50 % of an age- and sex-matched child [8].
Murine INCL The first PPT1-deficient mouse model was created by disrupting the CLN1 gene at the active site of PPT1 [9]. There are now two PPT1-deficient mouse models, both of which recapitulate the human disease and its natural progression [9,10]. PPT1-deficient mice have a shortened lifespan of ∼8.5 months, accumulate autofluorescent material and have widespread neurodegeneration, all of which are hallmarks of INCL. There is significant cortical thinning and overall brain atrophy of ∼30–40 %. Similar to human disease symptoms, PPT1-deficient mice display retinal degeneration and dysfunction, loss of motor co-ordination, and spontaneous seizures [11–13]. The PPT1-deficient mice also exhibit systemic disease such as left ventricular hypertrophy [14]. However, the progressive neurological disease has been the most thoroughly characterized. Within 3 months, focal astrocyte activation, visualized by glial fibrillary acidic protein (GFAP) up-regulation, is observed in the thalamus, cortex and cerebellum [11,15]. However, there is little change in clinical behaviour. By 5 months, there is significant neuronal loss in the CNS accompanied by a relatively rapid decline in motor function [11]. Finally, by 7–8 months, PPT1-deficient animals have severe motor impairment, spontaneous seizures, and a significant increase in microglial activation and macrophage infiltration [9,15–17]. Interestingly, astrocyte activation is the first histological sign of INCL and continues to worsen as the disease progresses [11]. Early reactive astrocytosis, visualized by GFAP immunoreactivity, appears to primarily affect astrocytes associated with blood vessels, potentially those involved in the blood–brain barrier [18]. However, as the mice age, the staining pattern shifts, and the distribution becomes more broad and indicative of hypertrophic processes.
filaments function to provide structural support, maintain axonal integrity and, under normal conditions, are typically not detectable by immunohistochemistry. Disruption of CNS function through a variety of insults results in a graded response of reactive astrogliosis. Though the role of reactive astrocytes in a diseased or traumatized brain is not fully understood, characterization of astrogliosis reveals astrocyte hypertrophy, changes in gene and protein expression, and modulation of inter- and intra-cellular signalling [22]. Up-regulation of intermediate filaments occurs in conjunction with hypertrophied astrocyte processes [23]. Histologically, severe astrocytosis may result in CNS restructuring and the formation of glial scars, particularly surrounding the area of insult. Owing to the interconnected network of astrocytes and neurons, CNS dysfunction may become exacerbated by astrogliosis due to functional deficits at synapses, blood–brain barrier breakdown and CNS metabolic defects [24]. In all, astrocytes provide a pivotal support structure for the CNS health and maintenance.
Astrocytosis and intermediate filaments Under normal conditions, GFAP and vimentin are expressed primarily in mature astrocytes, whereas vimentin and nestin are more highly expressed in immature astrocytes [20,25]. However, during CNS insult and reactive astrocytosis, there is up-regulation of the neurofilaments GFAP, vimentin and nestin [25]. Prior studies have indicated that loss of one neurofilament does not grossly disrupt intermediate filament formation [25]. However, deficiency in two neurofilaments (e.g. GFAP and vimentin) results in a complete deficiency of intermediate filament formation and a subsequent attenuation of astrocyte reactivity [26]. The loss of GFAP and vimentin reveal a dual role of reactive astrocytosis: a constructive role (e.g. wound healing, promoting neurite outgrowths, synaptic regeneration) as well as a destructive role (e.g. restricts neurogenesis, increases pathological vascularization) [22,27]. The loss of GFAP and vimentin appear to only have an effect when the CNS is challenged by trauma or aging [26]. Given the dual role of reactive astrocytosis, the focal areas of GFAP immunoreactivity early in INCL disease progression (before neuronal loss) raise the question of whether astrogliosis is beneficial or detrimental in INCL.
Astrocytes and astrocytosis Under healthy conditions (i.e. no trauma or neurodegeneration), astrocytes function to provide a supporting role to neurons and other CNS cell types [19,20]. Astrocytes function at synapses by maintaining osmolality, pH and neurotransmitter homoeostasis. In addition to its interaction with neurons, astrocytes also interact with neighbouring astrocytes and blood vessels [20]. This allows for a concerted regulation of local blood flow in the CNS and modulation of CNS metabolism [21]. A single astrocyte branches considerably and interacts with multiple neurons and synapses forming a non-overlapping domain with adjacent astrocytes [20]. Astrocytic branching is due in part to the expression of intermediate filaments. These intermediate
Astrocytosis in INCL Because astrocyte activation is one of the first pathological signs of disease observed in the PPT1-deficient brain, it is of particular interest to understand the role of reactive astrocytes in INCL disease progression. A murine model of attenuated astrocytosis was created by deleting both GFAP and vimentin [26]. One method to study the role of astrocytosis in INCL pathogenesis is to cross the PPT1 mutation model with a model of reactive astrocyte attenuation (GFAP-, vimentindeficient). The GFAP-, vimentin-, PPT1-deficient model (triple-knockout) showed an exacerbated clinical and C The
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pathophysiological phenotype as compared with the PPT1deficient model, although the hallmarks of INCL remained similar [18]. Perhaps the most dramatic finding was that the triple-knockout model resulted in a significantly shortened median lifespan (∼23 weeks) compared with the PPT1deficient model (∼33 weeks). Gross pathological examination revealed that the brain mass of triple-knockout mice was significantly reduced (∼30 %) compared with the PPT1deficient model (∼22 %). There was a significant decrease in cortical thickness and a significant increase in autofluorescent material in the triple-knockout mice compared with the PPT1-deficient mouse. The triple-knockout mice also have more widespread and rapidly progressing neurodegeneration observed by silver staining. Attenuated astrocytosis, due to the lack of GFAP and vimentin, leads to a significant exacerbation of the diseased state such that the 7-month-old PPT1deficient brain is comparable histologically with the 5-monthold triple-knockout brain. These data strongly suggest that the up-regulation of intermediate filaments plays a significant, potentially protective, role in INCL disease progression [18]. To determine the physiological role of astrocytosis in INCL, the authors evaluated the integrity of the blood–brain barrier and measured cytokine levels [18]. Analysis of the blood–brain barrier using an Evan’s Blue assay revealed that there was no gross perturbation to the barrier. Although the astrocytes may not be able to react appropriately to INCL disease pathogenesis, their functional role, at least in barrier support, was not inhibited. However, there appeared to be an increase in CD45 + leucocytes in the triple-knockout brain as compared with the PPT1-deficient model. Further analysis suggested that the leucocytes were activated microglia and monocytes/macrophages (CD68 + cells), as well as infiltrating CD3 + T-cells. These data further corroborate the positive role of reactive astrocytosis in regulating the extent of microglial activation and monocyte infiltration. The neuroinflammatory component is largely regulated by cytokine/chemokine levels. Therefore the cytokine levels in the triple-knockout were measured. Interestingly, cytokine levels across the board were significantly elevated at the 3-month time-point in the triple-knockout compared with either wild-type or PPT1-deficient animals. However, by 6 months of age, many of these differences had disappeared and the cytokine levels were comparable between tripleknockout mice and PPT1-deficient mice. The elevated cytokine levels at 3 months correlate with both the increased immune cell infiltration and the accelerated clinical disease course. These data demonstrate a role for astrocytes in the proper regulation of the neuroinflammatory response. Clearly, the lack of intermediate filaments and an attenuated astrocyte response is detrimental in INCL [18].
Concluding remarks Prior studies in both INCL and other NCL subtypes have shown significant glial activation before neuronal loss [e.g. the thalamacorticol pathology in the ceroid lipofuscinosis, neuroral 8 (CLN8)-deficient model], although C The
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whether the astrocytosis was reactive or causative was not examined [28–31]. Two prior studies showed that there was up-regulation of immune-system genes and mediators of neuroinflammation early in the disease progression, even before immunohistological evidence [10,17]. Macauley et al. [18] clearly show that astrocyte activation plays a prominent and protective role in INCL. Another study demonstrates further the importance of the neuroinflammatory component in INCL. In an effort to dissect the neuroinflammatory component of INCL, the PPT1-deficient mouse model was crossed with the Rag1-deficient mouse model (T- and B-cell deficient) [32]. The Rag1-, PPT1-deficient model revealed that the elimination of lymphocytes extended the lifespan and improved the clinical outcome [32]. This is consistent with the observation that astrocyte attenuation led to increased Tcell infiltration in the brains of triple-knockout mice and a dramatically accelerated disease phenotype [18]. Both Macauley et al. [18] and Groh et al. [32] showed the complicated interplay of reactive astrocytosis and the accompanying neuroinflammatory response. The neuroinflammatory component may be a significant contributor in other LSDs. For example, neuroinflammation has been shown to play an important role in the CNS disease associated with Gaucher’s disease [33], mucopolysacchridosis VII [34] and multiple sulfatase deficiency [35]. By identifying the mechanism bridging astrocytosis and neuroinflammation, we may begin to understand this complex interaction within the diseased brain and identify targets for therapeutics, such as utilizing novel drugs to counteract the neuroinflammatory component. Although such treatments will not correct the primary genetic defect, these approaches may be beneficial by lessening the severity of the neuroinflammatory response and act in concert with treatments designed to target the primary enzyme deficiency [36,37].
Acknowledgements We thank Dr Shannon Macauley and Dr Milos Pekny for input and discussion.
Funding This work was supported by the National Institutes of Health [grant number R01NS043205].
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22 Pekny, M., Wilhelmsson, U. and Pekna, M. (2014) The dual role of astrocyte activation and reactive gliosis. Neurosci. Lett. 565, 30–38 CrossRef PubMed 23 Pekny, M. and Pekna, M. (2004) Astrocyte intermediate filaments in CNS pathologies and regeneration. J. Pathol. 204, 428–437 CrossRef PubMed 24 Hoke, A. and Silver, J. (1994) Heterogeneity among astrocytes in reactive gliosis. Perspect. Dev. Neurobiol. 2, 269–274 PubMed 25 Eliasson, C., Sahlgren, C., Berthold, C.H., Stakeberg, J., Celis, J.E., Betsholtz, C., Eriksson, J.E. and Pekny, M. (1999) Intermediate filament protein partnership in astrocytes. J. Biol. Chem. 274, 23996–24006 CrossRef PubMed 26 Pekny, M., Johansson, C.B., Eliasson, C., Stakeberg, J., Wallen, A., Perlmann, T., Lendahl, U., Betsholtz, C., Berthold, C.H. and Frisen, J. (1999) Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin. J. Cell Biol. 145, 503–514 CrossRef PubMed 27 Li, L., Lundkvist, A., Andersson, D., Wilhelmsson, U., Nagai, N., Pardo, A.C., Nodin, C., Ståhlberg, A., Aprico, K., Larsson, K. et al. (2008) Protective role of reactive astrocytes in brain ischemia. J. Cereb. Blood Flow Metab. 28, 468–481 CrossRef PubMed 28 Kuronen, M., Lehesjoki, A.E., Jalanko, A., Cooper, J.D. and Kopra, O. (2012) Selective spatiotemporal patterns of glial activation and neuron loss in the sensory thalamocortical pathways of neuronal ceroid lipofuscinosis 8 mice. Neurobiol. Dis. 47, 444–457 CrossRef PubMed 29 Mirza, M., Volz, C., Karlstetter, M., Langiu, M., Somogyi, A., Ruonala, M.O., Tamm, E.R., Jagle, ¨ H. and Langmann, T. (2013) Progressive retinal degeneration and glial activation in the CLN6nclf mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation. PLoS ONE 8, e75963 CrossRef PubMed 30 Pontikis, C.C., Cotman, S.L., MacDonald, M.E. and Cooper, J.D. (2005) Thalamocortical neuron loss and localized astrocytosis in the Cln3ex7/8 knock-in mouse model of Batten disease. Neurobiol. Dis. 20, 823–836 CrossRef PubMed 31 Schmiedt, M.L., Blom, T., Blom, T., Kopra, O., Wong, A., von Schantz-Fant, C., Ikonen, E., Kuronen, M., Jauhiainen, M., Cooper, J.D. and Jalanko, A. (2012) Cln5-deficiency in mice leads to microglial activation, defective myelination and changes in lipid metabolism. Neurobiol. Dis. 46, 19–29 CrossRef PubMed 32 Groh, J., Kuhl, T.G., Ip, C.W., Nelvagal, H.R., Sri, S., Duckett, S., Mirza, M., Langmann, T., Cooper, J.D. and Martini, R. (2013) Immune cells perturb axons and impair neuronal survival in a mouse model of infantile neuronal ceroid lipofuscinosis. Brain 136, 1083–1101 CrossRef PubMed 33 Vitner, E.B., Farfel-Becker, T., Eilam, R., Biton, I. and Futerman, A.H. (2012) Contribution of brain inflammation to neuronal cell death in neuronopathic forms of Gaucher’s disease. Brain 135, 1724–1735 CrossRef PubMed 34 Parente, M.K., Rozen, R., Cearley, C.N. and Wolfe, J.H. (2012) Dysregulation of gene expression in a lysosomal storage disease varies between brain regions implicating unexpected mechanisms of neuropathology. PLoS ONE 7, e32419 CrossRef PubMed 35 Di Malta, C., Fryer, J.D., Settembre, C. and Ballabio, A. (2012) Astrocyte dysfunction triggers neurodegeneration in a lysosomal storage disorder. Proc. Natl. Acad. Sci. U.S.A. 109, E2334–E2342 CrossRef PubMed 36 Hawkins-Salsbury, J.A., Cooper, J.D. and Sands, M.S. (2013) Pathogenesis and therapies for infantile neuronal ceroid lipofuscinosis (infantile CLN1 disease). Biochim. Biophys. Acta 1832, 1906–1909 CrossRef PubMed 37 Hawkins-Salsbury, J.A., Reddy, A.S. and Sands, M.S. (2011) Combination therapies for lysosomal storage disease: is the whole greater than the sum of its parts? Hum. Mol. Genet. 20, R54–R60 CrossRef PubMed Received 8 July 2014 doi:10.1042/BST20140188
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Astrocytosis in infantile neuronal ceroid lipofuscinosis: friend or foe? Charles Shyng* and Mark S. Sands*†1 *Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A. †Department of Genetics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
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Abstract Infantile neuronal ceroid lipofuscinosis (INCL; infantile Batten disease) is an inherited paediatric neurodegenerative disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1) and is thus classified as a lysosomal storage disease. Pathological examination of both human and murine INCL brains reveals progressive, widespread neuroinflammation. In fact, astrocyte activation appears to be the first histological sign of disease. However, the role of astrocytosis in INCL was poorly understood. The hallmark of astrocyte activation is the up-regulation of intermediate filaments, such as glial fibrillary acidic protein (GFAP) and vimentin. The role of astrocytosis in INCL was studied in a murine model lacking PPT1 and the intermediate filaments GFAP and vimentin (triple-knockout). This murine model of INCL with attenuated astrocytosis had an exacerbated pathological and clinical phenotype. The triple-knockout mouse had a significantly shortened lifespan, and accelerated cellular and humoural neuroinflammatory response compared with the parental PPT1 − / − mouse. The data obtained from the triple-knockout mouse strongly suggest that astrocyte activation plays a beneficial role in early INCL disease progression. A more thorough understanding of the glial responses to lysosomal enzyme deficiencies and the accumulation of undergraded substrates will be crucial to developing effective therapeutics.
Lysosomal storage diseases Lysosomal storage disorders (LSDs) are a group of inherited metabolic diseases typically caused by a malfunction in the lysosome. Most LSDs are inherited in an autosomal recessive fashion and are due to deficiencies in lysosomal enzymes. Deficiencies in lysosomal enzyme activity result in the accumulation of non-degraded or partially degraded macromolecules. Although most storage accumulation is a direct result of aberrant or complete lack of enzyme activity, mutations to genes coding transport proteins and posttranslational modifiers can also result in an LSD phenotype [1]. The progressive accumulation of storage material is associated with a broad spectrum of cellular and clinical manifestations, such as organomegaly, neurodegeneration, skeletal dysplasia, lipid accumulation and developmental defects. Although each LSD has its own unique set of clinical symptoms, approximately two-thirds of LSDs have a neurological component [2].
Neuronal ceroid lipofuscinoses The neuronal ceroid lipofuscinoses (NCLs), or Batten disease, are a subset of LSDs caused by mutations to at least 13 different genes. Classified as the most common Key words: astrocytosis, glial fibrillary acidic protein, infantile neuronal ceroid lipofuscinosis, intermediate filament, lysosomal storage disease, vimentin. Abbreviations: CNS, central nervous system; GFAP, glial fibrillary acidic protein; INCL, infantile neuronal ceroid lipofuscinosis; LSD, lysosomal storage disorder; NCL, neuronal ceroid lipofuscinosis; PPT1, palmitoyl-protein thioesterase-1. 1 To whom correspondence should be addressed (email [email protected]).
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inherited paediatric neurological disorder (∼1 in 12 500 live births), NCL patients present with visual deficits, loss of motor co-ordination, severe cognitive decline and seizures [3]. Pathologically, the NCLs are defined by the accumulation of autofluorescent material in many cells of the body, but may be most pronounced in the brain [4]. Though the NCLs share clinical and histological presentations, particularly the progressive deterioration of the central nervous system (CNS), individual NCLs are distinct and are differentiated by genetic mutation, age, severity and ultrastructural characterization of storage material [5].
Infantile NCL Infantile neuronal ceroid lipofuscinosis (INCL; infantile Batten disease) is the earliest onset and most aggressive form of the NCLs. INCL is caused by an array of mutations in the CLN1 gene that lead to a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase1 (PPT1) [6]. The normal role of PPT1 is to cleave palmitoyl fatty-acyl chains from modified cysteine residues. Deficiency in PPT1 leads to the accumulation of the hallmark autofluorescent storage material, characterized ultrastructurally as granular osmiophilic deposits (GRODs). This accumulation is prevalent in all cell types, though it appears to predominate in the CNS [7]. The clinical presentation of INCL is similar to that of other NCLs, with the first clinical sign being blindness, followed by cognitive decline, motor dysfunction and intractable seizures. Initially, Biochem. Soc. Trans. (2014) 42, 1282–1285; doi:10.1042/BST20140188
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the patients develop normally and symptoms present between 6 and 12 months of age. By 3–4 years of age, patients typically enter a vegetative state and death occurs between 4 and 6 years of age. Autopsy reveals that INCL children have severe cortical atrophy with the brain mass equal to ∼50 % of an age- and sex-matched child [8].
Murine INCL The first PPT1-deficient mouse model was created by disrupting the CLN1 gene at the active site of PPT1 [9]. There are now two PPT1-deficient mouse models, both of which recapitulate the human disease and its natural progression [9,10]. PPT1-deficient mice have a shortened lifespan of ∼8.5 months, accumulate autofluorescent material and have widespread neurodegeneration, all of which are hallmarks of INCL. There is significant cortical thinning and overall brain atrophy of ∼30–40 %. Similar to human disease symptoms, PPT1-deficient mice display retinal degeneration and dysfunction, loss of motor co-ordination, and spontaneous seizures [11–13]. The PPT1-deficient mice also exhibit systemic disease such as left ventricular hypertrophy [14]. However, the progressive neurological disease has been the most thoroughly characterized. Within 3 months, focal astrocyte activation, visualized by glial fibrillary acidic protein (GFAP) up-regulation, is observed in the thalamus, cortex and cerebellum [11,15]. However, there is little change in clinical behaviour. By 5 months, there is significant neuronal loss in the CNS accompanied by a relatively rapid decline in motor function [11]. Finally, by 7–8 months, PPT1-deficient animals have severe motor impairment, spontaneous seizures, and a significant increase in microglial activation and macrophage infiltration [9,15–17]. Interestingly, astrocyte activation is the first histological sign of INCL and continues to worsen as the disease progresses [11]. Early reactive astrocytosis, visualized by GFAP immunoreactivity, appears to primarily affect astrocytes associated with blood vessels, potentially those involved in the blood–brain barrier [18]. However, as the mice age, the staining pattern shifts, and the distribution becomes more broad and indicative of hypertrophic processes.
filaments function to provide structural support, maintain axonal integrity and, under normal conditions, are typically not detectable by immunohistochemistry. Disruption of CNS function through a variety of insults results in a graded response of reactive astrogliosis. Though the role of reactive astrocytes in a diseased or traumatized brain is not fully understood, characterization of astrogliosis reveals astrocyte hypertrophy, changes in gene and protein expression, and modulation of inter- and intra-cellular signalling [22]. Up-regulation of intermediate filaments occurs in conjunction with hypertrophied astrocyte processes [23]. Histologically, severe astrocytosis may result in CNS restructuring and the formation of glial scars, particularly surrounding the area of insult. Owing to the interconnected network of astrocytes and neurons, CNS dysfunction may become exacerbated by astrogliosis due to functional deficits at synapses, blood–brain barrier breakdown and CNS metabolic defects [24]. In all, astrocytes provide a pivotal support structure for the CNS health and maintenance.
Astrocytosis and intermediate filaments Under normal conditions, GFAP and vimentin are expressed primarily in mature astrocytes, whereas vimentin and nestin are more highly expressed in immature astrocytes [20,25]. However, during CNS insult and reactive astrocytosis, there is up-regulation of the neurofilaments GFAP, vimentin and nestin [25]. Prior studies have indicated that loss of one neurofilament does not grossly disrupt intermediate filament formation [25]. However, deficiency in two neurofilaments (e.g. GFAP and vimentin) results in a complete deficiency of intermediate filament formation and a subsequent attenuation of astrocyte reactivity [26]. The loss of GFAP and vimentin reveal a dual role of reactive astrocytosis: a constructive role (e.g. wound healing, promoting neurite outgrowths, synaptic regeneration) as well as a destructive role (e.g. restricts neurogenesis, increases pathological vascularization) [22,27]. The loss of GFAP and vimentin appear to only have an effect when the CNS is challenged by trauma or aging [26]. Given the dual role of reactive astrocytosis, the focal areas of GFAP immunoreactivity early in INCL disease progression (before neuronal loss) raise the question of whether astrogliosis is beneficial or detrimental in INCL.
Astrocytes and astrocytosis Under healthy conditions (i.e. no trauma or neurodegeneration), astrocytes function to provide a supporting role to neurons and other CNS cell types [19,20]. Astrocytes function at synapses by maintaining osmolality, pH and neurotransmitter homoeostasis. In addition to its interaction with neurons, astrocytes also interact with neighbouring astrocytes and blood vessels [20]. This allows for a concerted regulation of local blood flow in the CNS and modulation of CNS metabolism [21]. A single astrocyte branches considerably and interacts with multiple neurons and synapses forming a non-overlapping domain with adjacent astrocytes [20]. Astrocytic branching is due in part to the expression of intermediate filaments. These intermediate
Astrocytosis in INCL Because astrocyte activation is one of the first pathological signs of disease observed in the PPT1-deficient brain, it is of particular interest to understand the role of reactive astrocytes in INCL disease progression. A murine model of attenuated astrocytosis was created by deleting both GFAP and vimentin [26]. One method to study the role of astrocytosis in INCL pathogenesis is to cross the PPT1 mutation model with a model of reactive astrocyte attenuation (GFAP-, vimentindeficient). The GFAP-, vimentin-, PPT1-deficient model (triple-knockout) showed an exacerbated clinical and C The
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pathophysiological phenotype as compared with the PPT1deficient model, although the hallmarks of INCL remained similar [18]. Perhaps the most dramatic finding was that the triple-knockout model resulted in a significantly shortened median lifespan (∼23 weeks) compared with the PPT1deficient model (∼33 weeks). Gross pathological examination revealed that the brain mass of triple-knockout mice was significantly reduced (∼30 %) compared with the PPT1deficient model (∼22 %). There was a significant decrease in cortical thickness and a significant increase in autofluorescent material in the triple-knockout mice compared with the PPT1-deficient mouse. The triple-knockout mice also have more widespread and rapidly progressing neurodegeneration observed by silver staining. Attenuated astrocytosis, due to the lack of GFAP and vimentin, leads to a significant exacerbation of the diseased state such that the 7-month-old PPT1deficient brain is comparable histologically with the 5-monthold triple-knockout brain. These data strongly suggest that the up-regulation of intermediate filaments plays a significant, potentially protective, role in INCL disease progression [18]. To determine the physiological role of astrocytosis in INCL, the authors evaluated the integrity of the blood–brain barrier and measured cytokine levels [18]. Analysis of the blood–brain barrier using an Evan’s Blue assay revealed that there was no gross perturbation to the barrier. Although the astrocytes may not be able to react appropriately to INCL disease pathogenesis, their functional role, at least in barrier support, was not inhibited. However, there appeared to be an increase in CD45 + leucocytes in the triple-knockout brain as compared with the PPT1-deficient model. Further analysis suggested that the leucocytes were activated microglia and monocytes/macrophages (CD68 + cells), as well as infiltrating CD3 + T-cells. These data further corroborate the positive role of reactive astrocytosis in regulating the extent of microglial activation and monocyte infiltration. The neuroinflammatory component is largely regulated by cytokine/chemokine levels. Therefore the cytokine levels in the triple-knockout were measured. Interestingly, cytokine levels across the board were significantly elevated at the 3-month time-point in the triple-knockout compared with either wild-type or PPT1-deficient animals. However, by 6 months of age, many of these differences had disappeared and the cytokine levels were comparable between tripleknockout mice and PPT1-deficient mice. The elevated cytokine levels at 3 months correlate with both the increased immune cell infiltration and the accelerated clinical disease course. These data demonstrate a role for astrocytes in the proper regulation of the neuroinflammatory response. Clearly, the lack of intermediate filaments and an attenuated astrocyte response is detrimental in INCL [18].
Concluding remarks Prior studies in both INCL and other NCL subtypes have shown significant glial activation before neuronal loss [e.g. the thalamacorticol pathology in the ceroid lipofuscinosis, neuroral 8 (CLN8)-deficient model], although C The
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whether the astrocytosis was reactive or causative was not examined [28–31]. Two prior studies showed that there was up-regulation of immune-system genes and mediators of neuroinflammation early in the disease progression, even before immunohistological evidence [10,17]. Macauley et al. [18] clearly show that astrocyte activation plays a prominent and protective role in INCL. Another study demonstrates further the importance of the neuroinflammatory component in INCL. In an effort to dissect the neuroinflammatory component of INCL, the PPT1-deficient mouse model was crossed with the Rag1-deficient mouse model (T- and B-cell deficient) [32]. The Rag1-, PPT1-deficient model revealed that the elimination of lymphocytes extended the lifespan and improved the clinical outcome [32]. This is consistent with the observation that astrocyte attenuation led to increased Tcell infiltration in the brains of triple-knockout mice and a dramatically accelerated disease phenotype [18]. Both Macauley et al. [18] and Groh et al. [32] showed the complicated interplay of reactive astrocytosis and the accompanying neuroinflammatory response. The neuroinflammatory component may be a significant contributor in other LSDs. For example, neuroinflammation has been shown to play an important role in the CNS disease associated with Gaucher’s disease [33], mucopolysacchridosis VII [34] and multiple sulfatase deficiency [35]. By identifying the mechanism bridging astrocytosis and neuroinflammation, we may begin to understand this complex interaction within the diseased brain and identify targets for therapeutics, such as utilizing novel drugs to counteract the neuroinflammatory component. Although such treatments will not correct the primary genetic defect, these approaches may be beneficial by lessening the severity of the neuroinflammatory response and act in concert with treatments designed to target the primary enzyme deficiency [36,37].
Acknowledgements We thank Dr Shannon Macauley and Dr Milos Pekny for input and discussion.
Funding This work was supported by the National Institutes of Health [grant number R01NS043205].
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