Charbel E.-H. Moussa

Amyloid precursor protein secretases as therapeutic targets for traumatic brain injury

Amyloid-β (Aβ) peptides, found in Alzheimers disease brain, accumulate rapidly after traumatic brain injury (TBI) in both humans and animals. Here we show that blocking either β- or γ-secretase, enzymes required for production of Aβ from amyloid precursor protein (APP), can ameliorate motor and cognitive deficits and reduce cell loss after experimental TBI in mice. Thus, APP secretases are promising targets for treatment of TBI.

Inflammation in the early stages of neurodegenerative pathology

Inflammation is secondary to protein accumulation in neurodegenerative diseases, including Alzheimers, Parkinsons and Amyotrophic Lateral Sclerosis. Emerging evidence indicate sustained inflammatory responses, involving microglia and astrocytes in animal models of neurodegeneration. It is unknown whether inflammation is beneficial or detrimental to disease progression and how inflammatory responses are induced within the CNS. Persistence of an inflammatory stimulus or failure to resolve sustained inflammation can result in pathology, thus, mechanisms that counteract inflammation are indispensable. Here we review studies on inflammation mediated by innate and adaptive immunity in the early stages of neurodegeneration and highlight important areas for future investigation.

Nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic degradation of α-synuclein in Parkinson's disease models

Parkinsons disease is a movement disorder characterized by death of dopaminergic substantia nigra (SN) neurons and brain accumulation of α-synuclein. The tyrosine kinase Abl is activated in neurodegeneration. Here, we show that lentiviral expression of α-synuclein in the mouse SN leads to Abl activation (phosphorylation) and lentiviral Abl expression increases α-synuclein levels, in agreement with Abl activation in PD brains. Administration of the tyrosine kinase inhibitor nilotinib decreases Abl activity and ameliorates autophagic clearance of α-synuclein in transgenic and lentiviral gene transfer models. Subcellular fractionation shows accumulation of α-synuclein and hyper-phosphorylated Tau (p-Tau) in autophagic vacuoles in α-synuclein expressing brains, but nilotinib enhances protein deposition into the lysosomes. Nilotinib is used for adult leukemia treatment and it enters the brain within US Food and Drug Administration approved doses, leading to autophagic degradation of α-synuclein, protection of SN neurons and amelioration of motor performance. These data suggest that nilotinib may be a therapeutic strategy to degrade α-synuclein in PD and other α-synucleinopathies.

Parkin mediates beclin-dependent autophagic clearance of defective mitochondria and ubiquitinated Aβ in AD models

Intraneuronal amyloid-β (Aβ) may contribute to extracellular plaque deposition, the characteristic pathology of Alzheimers disease (AD). The E3-ubiquitin ligase parkin ubiquitinates intracellular proteins and induces mitophagy. We previously demonstrated that parkin reduces Aβ levels in lentiviral models of intracellular Aβ. Here we used a triple transgenic AD (3xTg-AD) mouse, which over-expresses APP(Swe), Tau(P301L) and harbor the PS1(M146V) knock-in mutation and found that lentiviral parkin ubiquitinated intracellular Aβ in vivo, stimulated beclin-dependent molecular cascade of autophagy and facilitated clearance of vesicles containing debris and defective mitochondria. Parkin expression decreased intracellular Aβ levels and extracellular plaque deposition. Parkin expression also attenuated caspase activity, prevented mitochondrial dysfunction and oxidative stress and restored neurotransmitter synthesis. Restoration of glutamate synthesis, which was independent of glial-neuronal recycling, depended on mitochondrial activity and led to an increase in γ-amino butyric acid levels. These data indicate that parkin may be used as an alternative strategy to reduce Aβ levels and enhance autophagic clearance of Aβ-induced defects in AD. Parkin-mediated clearance of ubiquitinated Aβ may act in parallel with autophagy to clear molecular debris and defective mitochondria and restore neurotransmitter balance.

BETA AMYLOID-INDEPENDENT ROLE OF AMYLOID PRECURSOR PROTEIN IN GENERATION AND MAINTENANCE OF DENDRITIC SPINES

Synapse loss induced by amyloid beta (Abeta) is thought to be a primary contributor to cognitive decline in Alzheimers disease. Abeta is generated by proteolysis of amyloid precursor protein (APP), a synaptic receptor whose physiological function remains unclear. In the present study, we investigated the role of APP in dendritic spine formation, which is known to be important for learning and memory. We found that overexpression of APP increased spine number, whereas knockdown of APP reduced spine density in cultured hippocampal neurons. This spine-promoting effect of APP required both the extracellular and intracellular domains of APP, and was accompanied by specific upregulation of the GluR2, but not the GluR1, subunit of AMPA receptors. In an in vivo experiment, we found that cortical layers II/III and hippocampal CA1 pyramidal neurons in 1 year-old APP-deficient mice had fewer and shorter dendritic spines than wild-type littermates. In contrast, transgenic mice overexpressing mutant APP exhibited increased spine density compared to control animals, though only at a young age prior to overaccumulation of soluble amyloid. Additionally, increased glutamate synthesis was observed in young APP transgenic brains, whereas glutamate levels were decreased and GABA levels were increased in APP-deficient mice. These results demonstrate that APP is important for promoting spine formation and is required for proper spine development.

Parkin promotes intracellular Aβ1–42 clearance

Alzheimers disease and Parkinsons disease are common neurodegenerative diseases that may share some underlying mechanisms of pathogenesis. Abeta(1-42) fragments are found intracellularly, and extracellularly as amyloid plaques, in Alzheimers disease and in dementia with Lewy Bodies. Parkin is an E3-ubiquitin ligase involved in proteasomal degradation of intracellular proteins. Mutations in parkin, which result in loss of parkin function, lead to early onset Parkinsonism. Here we tested whether the ubiquitin ligase activity of parkin could lead to reduction in intracellular human Abeta(1-42). Lentiviral constructs encoding either human parkin or human Abeta(1-42) were used to infect M17 neuroblastoma cells. Parkin expression resulted in reduction of intracellular human Abeta(1-42) levels and protected against its toxicity in M17 cells. Co-injection of lentiviral constructs into control rat primary motor cortex demonstrated that parkin co-expression reduced human Abeta(1-42) levels and Abeta(1-42)-induced neuronal degeneration in vivo. Parkin increased proteasomal activity, and proteasomal inhibition blocked the effects of parkin on reducing Abeta(1-42) levels. Incubation of Abeta(1-42) cell lysates with ubiquitin, in the presence of parkin, demonstrated the generation of Abeta-ubiquitin complexes. These data indicate that parkin promotes ubiquitination and proteasomal degradation of intracellular Abeta(1-42) and demonstrate a protective effect in neurodegenerative diseases with Abeta deposits.

Parkin Protects against Mitochondrial Toxins and β-Amyloid Accumulation in Skeletal Muscle Cells

Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the β-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked β-amyloid precursor protein and for its metabolic byproducts Aβ42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular Aβ. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed β-amyloid. Correspondingly, exogenous Parkin significantly lowered Aβ levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of Aβ levels.

Parkin ubiquitinates Tar-DNA binding protein-43 (TDP-43) and promotes its cytosolic accumulation via interaction with histone deacetylase 6 (HDAC6).

Background: TDP-43 pathology and the role of E3 ubiquitin ligases are increasingly recognized in neurodegeneration. Results: Parkin ubiquitinates TDP-43 and forms a multiprotein complex with HDAC6 to sequester TDP-43 in cytosol. Conclusion: Parkin E3 ubiquitin ligase activity promotes TDP-43 inclusion formation and nuclear translocation. Significance: Parkin-TDP-43 interaction may be exploited as a therapeutic strategy in ALS/FTLD pathology. The importance of E3 ubiquitin ligases, involved in the degradation of misfolded proteins or promotion of protein-protein interaction, is increasingly recognized in neurodegeneration. TDP-43 is a predominantly nuclear protein, which regulates the transcription of thousands of genes and binds to mRNA of the E3 ubiquitin ligase Parkin to regulate its expression. Wild type and mutated TDP-43 are detected in ubiquitinated forms within the cytosol in several neurodegenerative diseases. We elucidated the mechanisms of TDP-43 interaction with Parkin using transgenic A315T mutant TDP-43 (TDP43-Tg) mice, lentiviral wild type TDP-43, and Parkin gene transfer rat models. TDP-43 expression increased Parkin mRNA and protein levels. Lentiviral TDP-43 increased the levels of nuclear and cytosolic protein, whereas Parkin co-expression mediated Lys-48 and Lys-63-linked ubiquitin to TDP-43 and led to cytosolic co-localization of Parkin with ubiquitinated TDP-43. Parkin and TDP-43 formed a multiprotein complex with HDAC6, perhaps to mediate TDP-43 translocation. In conclusion, Parkin ubiquitinates TDP-43 and facilitates its cytosolic accumulation through a multiprotein complex with HDAC6.

Tyrosine kinase inhibition increases functional parkin‐Beclin‐1 interaction and enhances amyloid clearance and cognitive performance

Tyrosine kinase inhibitors (TKIs) are effective therapies for leukaemia. Alzheimer is a neurodegenerative disease characterized by accumulation of β‐amyloid (plaques) and hyper‐phosphorylated Tau (tangles). Here we show that AD animals have high levels of insoluble parkin and decreased parkin‐Beclin‐1 interaction, while peripheral administration of TKIs, including Nilotinib and Bosutinib, increases soluble parkin leading to amyloid clearance and cognitive improvement. Blocking Beclin‐1 expression with shRNA or parkin deletion prevents tyrosine kinase (TK) inhibition‐induced amyloid clearance, suggesting that functional parkin‐Beclin‐1 interaction mediates amyloid degradation. Isolation of autophagic vacuoles (AVs) in AD mouse brain shows accumulation of parkin and amyloid, consistent with previous results in AD brains, while Bosutinib and Nilotinib increase parkin‐Beclin‐1 interaction and result in protein deposition in the lysosome. These data suggest that decreased parkin solubility impedes parkin‐Beclin‐1 interaction and amyloid clearance. We identified two FDA‐approved anti‐cancer drugs as potential treatment for AD.

β-Amyloid1–42 Gene Transfer Model Exhibits Intraneuronal Amyloid, Gliosis, Tau Phosphorylation, and Neuronal Loss

Alzheimer disease is characterized by extracellular β-amyloid (Aβ) plaques and intracellular inclusions containing neurofibrillary tangles of phospho-Tau and intraneuronal Aβ associated with neuronal cell death. We generated a novel gene transfer animal model using lentiviral Aβ1–42 that resulted in intracellular but not extracellular Aβ accumulations in the targeted rat primary motor cortex. Expression of intracellular Aβ1–42 led to pathological changes seen in human Alzheimer disease brains, including cell death, inflammatory signs, activation of two Tau kinases, and Tau hyperphosphorylation. Promoting clearance of lentiviral Aβ1–42 reversed these effects, demonstrating that intraneuronal Aβ1–42 is a toxic peptide that lies upstream of Tau modification. These studies reveal the role of intracellular Aβ1–42 in a novel gene transfer animal model, which is a useful tool to study intraneuronal Aβ1–42-induced pathology in the absence of extracellular plaques. Targeted delivery of Aβ will allow speedy delineation of pathological mechanisms associated with specific neurodegenerative lesions.