RESUMO
The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.
Assuntos
Encéfalo/fisiopatologia , Neuroglia/fisiologia , Lipofuscinoses Ceroides Neuronais/fisiopatologia , Neurônios/fisiologia , Adulto , Aminopeptidases/deficiência , Aminopeptidases/genética , Animais , Encéfalo/patologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Criança , Técnicas de Cocultura , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Dipeptidil Peptidases e Tripeptidil Peptidases/deficiência , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Feminino , Glutationa/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genética , Neuroglia/patologia , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/patologia , Serina Proteases/deficiência , Serina Proteases/genética , Tripeptidil-Peptidase 1 , Adulto JovemRESUMO
The ability to reliably quantify the relative degree of storage burden that results from lysosomal dysfunction is an important goal. Such measurements not only allow an assessment of different stages of disease progression, but also the assessment of therapeutic strategies. Although biochemical methods exist for doing this, retaining the anatomical integrity of tissue samples is an important consideration. This chapter provides practical methodological recommendations for achieving this goal in tissue sections, either by directly visualizing or staining the storage material, and subsequently quantifying it via image analysis.
