RESUMO
Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Adulto , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Atividade Motora/fisiologia , Neurônios Motores/patologia , Mutação , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Adulto JovemRESUMO
The development of therapeutics for neurological disorders is constrained by limited access to the central nervous system (CNS). ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed on the luminal surface of capillaries in the CNS and transport drugs out of the endothelium back into the blood against the concentration gradient. Survival motor neuron (SMN) protein, which is deficient in spinal muscular atrophy (SMA), is a target of the ubiquitin proteasome system. Inhibiting the proteasome in a rodent model of SMA with bortezomib increases SMN protein levels in peripheral tissues but not the CNS, because bortezomib has poor CNS penetrance. We sought to determine if we could inhibit SMN degradation in the CNS of SMA mice with a combination of bortezomib and the ABC transporter inhibitor tariquidar. In cultured cells we show that bortezomib is a substrate of P-gp. Mass spectrometry analysis demonstrated that intraperitoneal co-administration of tariquidar increased the CNS penetrance of bortezomib, and reduced proteasome activity in the brain and spinal cord. This correlated with increased SMN protein levels and improved survival and motor function of SMA mice. These findings show that CNS penetrance of treatment for this neurological disorder can be improved by inhibiting drug efflux at the blood-brain barrier.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , Bortezomib/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Antineoplásicos/farmacologia , Sistema Nervoso Central/citologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Fatores de Tempo , TransfecçãoRESUMO
Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.
Assuntos
Esclerose Lateral Amiotrófica/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Splicing de RNA , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Proteínas de Ligação a DNA/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , UbiquitinaçãoRESUMO
Spinal muscular atrophy (SMA) is a debilitating neurological disorder marked by degeneration of spinal motor neurons and muscle atrophy. SMA results from mutations in survival motor neuron 1 (SMN1), leading to deficiency of survival motor neuron (SMN) protein. Current therapies increase SMN protein and improve patient survival but have variable improvements in motor function, making it necessary to identify complementary strategies to further improve disease outcomes. Here, we perform a genome-wide RNAi screen using a luciferase-based activity reporter and identify genes involved in regulating SMN gene expression, RNA processing, and protein stability. We show that reduced expression of Transcription Export complex components increases SMN levels through the regulation of nuclear/cytoplasmic RNA transport. We also show that the E3 ligase, Neurl2, works cooperatively with Mib1 to ubiquitinate and promote SMN degradation. Together, our screen uncovers pathways through which SMN expression is regulated, potentially revealing additional strategies to treat SMA.
Assuntos
Técnicas Genéticas/normas , Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Neurônios Motores/metabolismo , Interferência de RNA/fisiologia , HumanosRESUMO
Autosomal-recessive proximal spinal muscular atrophy (Werdnig-Hoffmann, Kugelberg-Welander) is caused by mutation of the SMN1 gene, and the clinical severity correlates with the number of copies of a nearly identical gene, SMN2. The SMN protein plays a critical role in spliceosome assembly and may have other cellular functions, such as mRNA transport. Cell culture and animal models have helped to define the disease mechanism and to identify targets for therapeutic intervention. The main focus for developing treatment has been to increase SMN levels, and accomplishing this with small molecules, oligonucleotides, and gene replacement has been quite. An oligonucleotide, nusinersen, was recently approved for treatment in patients, and confirmatory studies of other agents are now under way.
Assuntos
Atrofia Muscular Espinal , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Humanos , Morfolinos/uso terapêutico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Mutação/genética , Oligonucleotídeos/uso terapêutico , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neurodegenerative disorder, and cytoplasmic inclusions containing transactive response (TAR) DNA binding protein (TDP-43) are present in ~90 % of cases. Here we report detailed pathology in human TDP-43 transgenic mice that recapitulate key features of TDP-43-linked ALS. RESULTS: Expression of human wild-type TDP-43 (TDP-43(WT)) caused no clinical or pathological phenotype, while expression of Q331K mutant (TDP-43(Q331K)) resulted in a non-lethal age-dependent motor phenotype, accompanied by cytoplasmic TDP-43 aggregation, mild neuronal loss, with astroglial and microglial activation in the motor cortex and spinal cord at 24 months. However, co-expression of WT and Q331K mutant (TDP-43(WTxQ331K)) resulted in an extremely aggressive motor phenotype with tremor from 3 weeks and progressive hind-limb paralysis necessitating euthanasia by 8-10 weeks of age. Neuronal loss and reactive gliosis was observed in the spinal cord and layer V region of the cortex, with TDP-43, ubiquitin and p62 cytoplasmic inclusions and an increase in insoluble TDP-43. Nuclear clearance of TDP-43 was not observed in TDP-43(Q331K) mice but was seen in 65 % of aggregate containing spinal cord motor neurons in TDP-43(WTxQ331K) mice. CONCLUSIONS: We hypothesise that cytoplasmic TDP-43(Q331K) aggregates facilitate the recruitment of WT protein in compound animals, which dramatically accelerates neurodegeneration and disease progression. The exploration of disease mechanisms in slow and rapid disease models of TDP-43 proteinopathy will help elucidate novel drug targets and provide a more informative platform for preclinical trials.