Alzheimer’s disease (AD) is characterized by profound central neurodegeneration. While it is a recurring theme in many neurological and neuro-psychiatric disorders, the precise mechanisms leading to the loss of neuronal mass have varying etiologies. In neurofibrillary degeneration of Alzheimer type, the affected neurons exhibit a particular type of pathology that evolves in a distal-to-proximal manner. This so-called, retrograde degeneration begins distally in the axo-dendritic network disrupting normal synaptic activity. The basis for retrograde degeneration can be explained by the presence of altered microtubule (MT) dynamics.
MTs play an essential role in maintaining the structural and physiological integrity of neurons. Neurodegenerative disorders, including AD, show evidence of cytoskeletal dysfunction at both cytoarchitectural and molecular levels. Changes in neuronal shape, loss of dendrites and dendritic spines and abnormal accumulation of cytoskeletal proteins either in the form of inclusions like neurofibrillary tangles of paired helical filaments (PHF) seen in AD [24
], or Lewy bodies seen in Parkinson’s disease [29
], constitute evidence of cytoarchitectural abnormalities. These neurons with anatomical aberrations show impaired function, which is seen as loss of synapses, decrease in synaptic conductivity and synaptic plasticity. This places the neuronal cytoskeleton as a key target for research and drug development.
In the normal brain, MT assembly is stimulated by a number of microtubule-associated proteins (MAP), the high molecular weight MAP1 and MAP2, as well as the 50–75 kDa tau proteins [37
]. It was previously reported by us that in AD, tau is abnormally hyperphosphorylated and aggregated into bundles of paired helical filaments (PHFs), which progressively replace the neuronal cytoskeleton [20
]. In situ and in vitro observations suggest that the cytosolic/non-fibrillized abnormally hyperphosphorylated tau is inhibitory to MT assembly and stability [1
], whereas fibrillized tau is not [5
]. Moreover, only hyperphosphorylated tau, and neither MAP1 nor MAP2, has been shown to accumulate in the affected neurons in AD and related tauopathies. While it is desirable to study the effects of tau hyperphosphorylation on MT function in an in vivo system, there are several limitations surrounding direct and isolated manipulation of tau phosphorylation and its effect on MT dynamics in the living cell. Both genetic and pharmacologic means of studying mechanisms of neurofibrillary degeneration have several pitfalls. Transfected cell lines and transgenic animals overexpressing wild type, pseudophosphorylated or mutated tau do not reflect the pathology in AD in which neither any causative tau gene mutation nor increase in tau messenger RNA has been found [15
]. Modifying kinase and phosphatase levels/activities in the cell to achieve tau hyperphosphorylation [13
] affects several other pathways in the cell [6
] and may not truly reflect the disease state. Consequently, the mechanisms proposed for the apparent toxicity of hyperphosphorylated tau have not yet been identified in the cell and therefore remain unestablished.
Detergent extraction of cells has been extensively employed in the literature to study components of the cytoskeleton [7
]. When cells are treated with a non-ionic detergent, such as Triton X-100, approximately 70% of the cytosolic proteins are extracted [30
]. Such detergent-extracted cells retain many of the morphological features of the cytoskeleton seen in intact cells, including microfilaments, microtubules, intermediate filaments and associated proteins [16
Here, we describe a model in which we disrupted the MT network of 3T3 cells (mouse embryonic fibroblasts) with Nocodazole (Noco), followed by removal of the cytoplasm by extraction with Triton X-100 (Tri), and replaced the MT network of the host cells (Noco/Tri cells) with that polymerized from adult rat brain cytosol. The MT network formed from rat brain tubulin, MAP2 and tau created a neuron-like environment in which we could directly study the effects of AD P-tau in real time. We found that AD P-tau causes breakdown of MT network by sequestering both tau and MAP2. A compromised MT network both in the axons and dendrites might be the basis of the progressive retrograde degeneration of the affected neurons seen in AD and related tauopathies.