amyloid peptides are derived from the amyloid precursor protein ( app ) by sequential proteolytic cleavages by - and -secretases [ 1 , 2 ] . amyloid peptides , particularly the longer a42 species , aggregate into various and different chemical or physical entities , which are now considered the primary pathological agents in ad . controversial experimental data leave nevertheless many questions open with regards to the exact composition and size of the amyloid oligomers in vivo [ 35 ] . moreover , the precise molecular actions of the amyloid peptides and their aggregates remain unknown and enigmatic , largely because their molecular targets , or their specific receptors remain undefined . this issue raises more fundamental questions . for one , while the proteolytic enzymes and their mechanisms responsible for the generation of the amyloid peptides become well - known and understood at the molecular level , the eventual physiological function of the peptides is still questioned  and remains questionable . the original pathological importance ascribed to amyloid plaques was weakened , if not eliminated based on information gathered in transgenic models expressing mutant app . we have advocated this hypothesis since we discovered early defects in cognition and ltp in app.v717i mice [ 611 ] . matters are , however , complicated further by recent data originating from several clinical trials that identified a large fraction of individuals in the control groups with considerable brain amyloid load using pet - imaging [ 1214 ] . obviously , this means that high - amyloid concentration in the brain is not per se incompatible with normal cognitive functioning in old age . this calls for other factors to be at least coresponsible for the cognitive demise in ad - forwarding protein tau as the most obvious candidate . primary tauopathy is recognized in a vast and still increasing number of cns - disorders . the pathology is visualized postmortem as intraneuronal aggregates of protein tau , known as neuropil treads and neurofibrillary tangles . these are similar if not identical to those observed in ad brain and mostly present without other associated pathological hallmarks , except inflammation markers . the large clinical variability among the primary tauopathies stems from the presence of the tau pathology in different types of neurons and in different brain - regions [ 1518 ] . biochemically , all tauopathies consist of smaller and larger aggregates and fibrils that become intertwined to form larger tangles in the soma as well as in axons and dendrites of affected neurons . the aggregates consist almost exclusively of protein tau , albeit as different isoforms in different diseases , that is , either tau.3r or tau.4r or as a variable admixture . their widely different grading of phosphorylation is usually referred to as hyperphosphorylated tau , denoted here as hp - tau . importantly , no accurate definition based on either the level or the specific phosphorylated residues can typify any tauopathy exactly , because of the large variability with each primary disease . . rare familial cases of frontotemporal dementia ( ftd ) are linked to exonic and intronic mutations in the mapt gene coding for protein tau ( chromosome 17 ) . although rare , their identification marked a major breakthrough and boosted interest in tauopathy , because of the evident implication that tauopathy itself is sufficient to cause cognitive decline and dementia [ 15 , 16 , 1821 ] . both intronic and exonic mutations have important mechanistical implications : ( i ) most expressed mutations are in the microtubule binding domain of tau ; ( ii ) intronic mutations promote or hinder splicing of exon 10 coding for the second repeat domain in the microtubule binding domain , skewing expression of isoforms to either tau3r or tau4r , but both with normal sequences and consequently normal binding - affinity for microtubules . the pathology resulting from intronic mutations in the mapt gene can only be explained by effects on rna splicing , eventually resulting in changes in absolute or relative levels of tau isoforms that otherwise have normal wild - type protein sequences . the overexpression or overrepresentation of one or the other tau isoform can then be extrapolated to the primary tauopathies that are sporadic , as well as to sporadic ad as the most prominent secondary tauopathy involving normal , wild - type tau - isoforms . the major unresolved problem in ad is the mechanistic relation of amyloid and tau pathology . amyloid oligomers are proposed to precede , and eventually trigger the intracellular tauopathy , likely by increasing the phosphorylation of protein tau [ 11 , 15 , 17 , 22 ] . while this hypothesis remains at this moment in time impossible to prove in sporadic ad cases , indeed , even in the most early onset familial ad that are caused by mutant presenilin or mutant app , and therefore by definition are  amyloid - triggered  , tauopathy is always evident and codiagnostic . experimentally , double and triple transgenic mouse models with combined amyloid and tau - pathology prove the same intimate relation [ 2326 ] . the genetic data imply that deranged tau - microtubule interactions , caused either directly by the mutation , by phosphorylation , by increased absolute or by disturbed relative concentrations of tau , all can contribute or even be sufficient to cause neurodegeneration in primary tauopathies , that is , in the absence of amyloid pathology . importantly , experimental decrease of the level of tau in amyloid transgenic mice can actually ameliorate several clinical symptoms and defects . the combined data further corroborate the hypothesis that protein tau is essentially contributing to amyloid - induced cognitive defects .   not resolved are the identity and exact nature of the receptors and associated signaling pathways that lead from amyloid to tau . these pathways obligatory involve kinases such as gsk3 [ 25 , 26 ] that contribute , directly or indirectly to the phosphorylation of tau at residues that affect its binding to microtubuli . this is thought , but not proven , to instigate its eventual aggregation into fibrils that are deposited locally as neuropil treads or transported to the soma and form the neurofibrillary tangles , and both types of aggregates occur in the same neurons . nevertheless , tauopathy does not per se cause immediate or even delayed neuronal death , as tangled and tau - loaded neurons are observed in humans as well as in experimental models , that is , transgenic mice , zebra - fish , and flies [ 26 , 28 , 29 ] . the pertinent question to be answered then is : what chemical or physical form of protein tau causes synapto- and neurodegeneration ? invaluable insight in the problems that we are concerned with , have resulted from analyzing different transgenic mouse models . our research - group generated , characterized , and validated many different transgenic mouse strains and their bigenic combinations as preclinical models over the last two decades . so far none of the models recapitulates robustly the evident neurodegeneration observed in the brain of ad patients . we explored and reported on additional models to increase our understanding of this aspect of the pathogenesis of ad [ 11 , 30 ] . here , we review data and insights obtained with adeno - associated viral vectors that were engineered to express wild - type or mutant app or protein tau in pyramidal neurons of the hippocampus of wild - type mice . thereby , we aimed to recapitulate aspects of ad pathology and related features that are not attainable in transgenic mice , particularly neurodegeneration . among the different viral vectors available , we selected aav because these have been demonstrated as excellent and safe tools for gene delivery into the cns . recent technical advances have improved aav - based vectors as tools in neuronal research [ 31 , 32 ] . the expression of the human transgenes used in our studies was controlled by the human synapsin1 gene promoter , with constructs packed in chimeric capsids aav1/2 , expressing efficiently in pyramidal neurons of the ca1/2 subfield of the hippocampus ( figure 1 ) . control aav vectors expressed egfp in the designated neurons and areas that persisted for months without negative signs or symptoms ( figure 1 ) and [ 31 , 32 ] . we first constructed aav vectors to recapitulate the amyloid pathology in the limbic region of wild - type mice . the constructs contained app695 as the most abundant neuronal isoform , either as wild type sequence or as the engineered triple mutant ( denoted app.sla ) . the incorporated swedish , london and austrian mutations are each associated with familial forms of ad [ 3335 ] .   following intracerebral injection into wild - type mice , the app.sla mutant produced a gradual accumulation of app and amyloid peptides in pyramidal neurons in the hippocampal ca1/2-region , as well as in the deeper layers of the neighboring cortex . similar expression of wild - type human app695 failed to produce any deposition of amyloid or of the related effects observed with mutant app.sla .   initially , from 3 weeks to 3 months postinjection ( p.i . ) , amyloid immunoreactivity appeared as intracellular inclusions or vacuolar bodies inside pyramidal neurons . at 6 months , amyloid plaques developed as immunoreactive and thioflavins positive deposits in the hippocampus and cortex ( figure 2 ) . at this late time - point , we also observed increased phosphorylation of endogenous mouse protein tau , particularly at residues t181 and t231 , both known to be substrate for gsk3 . these phosphoepitopes , defined by antibodies at270 and at180 were differentially expressed , that is , in hippocampal pyramidal neurons ( t231 ) and in dystrophic neurites around amyloid plaques ( t181 ) . in contrast , intracerebral injection of aav-app.sla not in wild - type but in tau.p301l mice led to the formation of neurofibrillary tangles ( nft ) , demonstrating that the viral assault did not prevent protein tau from forming tangles ( figure 2 ) . moreover , the combination demonstrated that besides the primary hit , also the receiving genetic background is of utmost importance . most interesting for the interpretation of the data obtained with protein tau , discussed in the next section , was the observation that at 6 months p.i . the phosphorylation of endogenous mouse tau was increased and coincided with a minor but significant reduction in the number of neurons in the ca1 region of aav-app.sla injected mice .   in second instance , we similarly expressed either wild - type tau.4r or mutant tau.p301l by aav - vectors in wild - type mouse brain . in sharp contrast to app - sla , aav - mediated expression of protein tau resulted already at 3 weeks p.i . these findings were rather unexpected and we analyzed them in different directions , yielding novel and important insight into the mechanisms causing neuronal death by protein tau . most remarkably , we could not detect any major form of aggregates of protein tau in pyramidal neurons that expressed either wild - type or mutant protein tau . extensive analysis by histochemistry and immunohistochemistry with a panel of indicator compounds and monoclonal antibodies to protein tau , did not reveal any appreciable signs of formation of intraneuronal tau - aggregates , either before or during or after the pyramidal neurons in ca1/2 succumbed . the actual levels in hippocampal extracts were near - physiological and only about twice those of endogenous mouse tau4r . note that also in the aav-app.sla model described in the previous section , the hippocampal levels of human app.sla were only about twice those of endogenous murine app . in addition , expression of egfp by the same aav - type vectors proved harmless to pyramidal neurons over long time - periods ( figure 1 ) and [ 30 , 31 ] . intracerebral injection of 3- and 10-fold less aav - tau , with consequently less expression of human protein tau , produced a graded , lesser loss of hippocampal neurons . the aav - tau - induced neurodegeneration is thereby further demonstrated to be directly caused by and proportional to the near - physiological levels of protein tau . consequently , the observed pyramidal cell - death is qualified as specific for human protein tau . this conclusion holds up for wild - type tau.4r and for mutant tau.p301l , making the model at least conform the observations in familial ftd . indeed , as discussed above , intronic and exonic mutations yield very similar clinical outcome , despite the expression of mutant or wild - type tau4r , respectively . the actual contribution of the mutation to the mechanism underlying the pathogenesis requires a rational explanation in human ftd patients , as well as in the aav - models . extensive analysis to define the mechanisms that underlie the observed tau - mediated pyramidal neurodegeneration are described , summarizing reported data and preliminary data of work in progress . similar aav - mediated expression of a truncated version of protein tau.4r , lacking the microtubule binding c - terminal domain , proved completely harmless and did not cause any neurodegeneration over similar time - periods . the inherent conclusion must be that microtubule binding of protein tau is essentially involved in the neurotoxic degenerative mechanism . consequently , we must orient further analysis to mechanisms that involve axonal and dendritic transport over microtubuli , which is of paramount importance for all cells but especially for neurons with their intricate branching .   of note , microgliosis was observed to be intense , and spatially and temporally closely associated with aav - tau induced degenerating neurons . this was most recently also reported in a similar model based on aav vectors but in rats and for a different pathology in a different brain - region . previously , we have observed a similar relation of microgliosis to neurodegeneration in an unrelated model for hippocampal sclerosis , caused by conditional expression of p25 , the truncated activator of cdk5 . also , in that model , intense neurodegeneration was not marked by aggregation of protein tau , further strengthening our conclusion that cdk5 is not a major tau - kinase in vivo [ 3941 ] . the data originating from the aav - models led us to conclude that not large aggregates of protein tau cause neurodegeneration . this confirms our observations in transgenic mice that show aggregation of tau leading to tauopathy in somata and neuropil , but not accompanied by marked neurodegeneration [ 25 , 26 , 36 ] . we proposed that neurons affected by tauopathy can either engage in aggregation of tau and thereby try to decrease the toxic species and hope to survive , or to enter the  path to death  by failing to aggregate protein tau . clearly , the viral models comply with the transgenic models that aggregation of tau and neurodegeneration are not closely linked . thereby , questions are raised that are addressed in more detail in the next sections .   the classic concept that tau - fibrils or tangles are neurotoxic in tauopathies can be abandoned . we advocate that neurofibrillary tangles , like amyloid plaques are the final pathological hallmarks , but not the neurotoxic agents . the outcome fits independent observations that the extent of neuronal loss exceeds the number of nft in patients with ad [ 42 , 43 ] . in inducible tg4510 mice , memory decline and neuronal loss are dissociated from tangle formation in time and brain region [ 44 , 45 ] .   in the aav - tau injected mice , phosphorylation of tau was evident at many pathological epitopes , including those defined by antibodies at8 , at100 , at180 , and at270 . despite the increased phosphorylation no aggregates of protein tau were deposited in degenerating pyramidal neurons , analyzed histochemically or immunohistochemically . biochemical analysis indicated the formation of low molecular weight aggregates   which need and deserve further analysis . intriguingly , experiments whereby aav-app.sla was injected intracerebrally not in wild - type mice but in transgenic tau.p301l mice , produced not only amyloid plaques but also intracellular tau aggregates . nevertheless , no substantial neuronal loss was evident , substantiating our previous data of absence of marked neurodegeneration in tau.p301l mice and tau.p301lxgsk3b bigenic mice ( bigt ) , despite extensive or even dramatic tauopathy [ 26 , 36 ] . the combined results from our experimental models and from a drosophila model of tauopathy   consolidates the thesis that neurotoxicity is not exerted by large aggregates of protein tau but rather by tau - species that are intermediate between normally phosphorylated protein tau and the hyper - phosphorylated fibrils . the identity of the toxic tau species , which we have termed  tau - p *  [ 11 , 30 ] remains to be defined  a challenge equaling that of the identification of the amyloid receptor . it must be remembered that tau is subject to other posttranslational modifications , besides phosphorylation . indeed , tau can be ubiquitinated , truncated , glycosylated , glycated , oxidized , and , moreover , undergoes isomerization at proline residues . at any given moment protein tau is present in various molecular forms , which most likely differ subtly in properties of binding to microtubuli , interaction with other proteins , binding to membranes , being transported for normal duty or for degradation . the decision whether neurons will enter the cell death path or protect itself by forming tauopathy , must depend on the actual levels of protein tau and on its modifications . the rapid accumulation of tau - p * will lead to cell death , while more gradual accumulation of tau - p * would result in formation of aggregates . both pathways will be affected by various external factors , for example , inflammation , amyloid , stress , and so forth . in this concept , evidence from patients , whereby ca1 hippocampal neurons survive for decades despite neurofibrillary tangles   and experimental animals show tangle bearing neurons survive , even with lost membrane integrity . two major pathways are responsible for removal of damaged , unfolded , or aggregated cellular proteins : the ubiquitin / proteasome system and the autophagy / lysosomal system that also can remove aged or damaged organelles . malfunction of autophagy can result in accumulation of protein aggregates that may contribute to the disease pathology in ad and other neurodegenerative disorders [ 4952 ] . stimulating autophagy pharmacologically , for example , by inhibition of mtor , ameliorates cognitive deficits and reduces amyloid and tau pathology in the 3xtg mouse model for ad . autophagosomes form in a stepwise process that requires transport over the microtubular network , which is known to be affected by protein tau , a major microtubule - associated protein .   in our mouse models , degenerating neurons in the aav - tau.p301l injected mice displayed as dark neurons with extensive vacuolization that could be taken to indicate a defective autophagic process . a similar facet of neurons we observed in an unrelated mouse model , that is , the p25 inducible mice that recapitulate hippocampal sclerosis . the combined data do lend more support to the hypothesis that vacuolization is a correlate or consequence of the degenerative process , as an intrinsic mechanism needed to clean up defective organelles , protein aggregates or even entire dead or dying neurons . consistent with this view is our observation in the aav - tau.p301l mice that vacuoles do not form early in the disease process but only after the neurons are already degenerating [ 11 , 30 ] . finally , we searched but did not find biochemical evidence for the conversion of lc3-i to lc3-ii , which is generally accepted as the necessary step in and read - out of the increased formation of autophagosomes . after differentiation from neuronal precursors , neurons enter their post - mitotic state , while attempts to reenter the cell cycle is considered pathological and eventually result in cell death . in ad and other tauopathies , cell - cycle related events we tested this hypothesis in the aav - tau model by examining cell cycle - related markers . various markers were increased , that is , cyclind2 , phosphorylated retinoblastoma protein , proliferating cell nuclear antigen ( pcna ) , cyclin b1 . however , intracerebral injection of aav - tau in cyclind2 deficient mice showed no extra effect on neurodegeneration , suggesting that cell cycle reentry is not a pre - requisite for neuronal death . microtubules ensure cell shape and constitute roads of transport , a feature prominent in neurons with the most complex architecture of all cells . microtubule dependent transport is ensured by families of motor proteins dyneins and kinesins , respectively for retrograde transport from distal processes towards soma and as plus - end directed motor for anterograde transport . first , mutant tau and hp - tau can cause it to detach from the microtubules and decrease its ability to control microtubule dynamics . on the other hand , increased levels of protein tau can saturate microtubules and hinder the  foot - stepping  of the motor proteins needed for axonal and dendritic transport . both aspects of tau - related transport deficits have been observed and both can fit into a model leading to  starving synapses  that eventually culminates in neuronal death . in this hypothesis , tau - mediated neurodegeneration is likely to start in distal neuronal processes , that is , axons and apical dendrites , which degenerate progressively towards the cell soma . synaptic pathology could be an early defect in neurodegenerative tauopathies   as proper functioning of distal synapses is totally dependent on adequate transport of essential cargos and uninterrupted provision of energy . impaired oxidative phosphorylation and mitochondrial dysfunction has been observed in mouse models expressing mutant tau , which was potentiated by mutant app and presenilin1 [ 56 , 57 ] .   in tau.4r transgenic mice , we previously documented axonopathy and motor deficits that were rescued by coexpression of constitutively active gsk3b [ 37 , 58 ] . the data explain how excess tau in first instance blocks transport by covering microtubuli , while gsk3 acting as major tau - kinase i phosphorylates tau to detach it from the microtubules and relieves the transport blockade . interestingly , treatment of tau.4r mice with lithium salts significantly increased the axonopathy , an effect that can be ascribed to its inhibition of gsk3b . as discussed above , aav - mediated expression of truncated tau , lacking the microtubule binding domain did not cause neurodegeneration , in marked contrast to full length protein tau ( figures 3 and 4 ) and . the combined data from various approaches allow us to conclude that tau mediated pathology is , at least in part , mediated by its microtubule binding properties . chronic inflammation is evident in brain of ad patients and proposed to contribute essentially to the  vicious circle  leading eventually to neuronal death , brain atrophy and severe dementia . cns - related features of the immune system remain hardly understood , and supposed to be  janus - faced  , that is , providing protection as well as causing damage , steered by complex mostly unknown control mechanisms [ 60 , 61 ] . protection could be impaired in ad , fueled by  and fueling  debates on possible prevention or therapeutic benefits of antioxidants , radical scavengers , nonsteroidal anti - inflammatory drugs ( nsaid ) , and so forth , although clinical trials do not substantiate the claims . amyloid deposits attract microglia in a phagosomal attempt of their elimination , but activated microglia release proinflammatory cytokines , chemokines , reactive oxygen species , prostaglandins , and other mediators that harm neurons . much attention has been paid to neurotoxicity mediated by free radicals , both reactive oxygen and nitrogen species , released by microglia . astrocytes are the most common cell - type in the brain , involved in many functions and increasingly appreciated also in synaptic signaling and integrity . astrocytes co - localize with microglia at damage - sites , but remain to be explored in detail in ad . strictly speaking , neurons must also be considered when discussing neuroinflammation because they ( can ) produce active factors , that is , complement , interleukins , tumor necrosis factor , acute - phase proteins . we observed and discussed that degenerating neurons were temporally and spatially closely associated with activated microglia in two independent models , that is , the aav - tau viral model and the p25 transgenic model [ 30 , 39 ] . too many  unknowns  prevent us from answering the question how tau - mediated damage can lead to neurodegeneration by involving activation of microglia . we maintain the most straightforward hypothesis , that is , that the expression of protein tau , either wild - type or mutant by the viral vectors , is imposing stress on the pyramidal neurons that can not adapt rapidly enough to produce tau - aggregates as a means of temporarily detoxification . the impact on various neuronal functions is evident , including disturbances in post - mitotic state and cell - cycle control , as well as disturbing microtubule - mediated transport . the latter is most destructive in axons and larger dendrites , compromising the major synapses that become dys- or nonfunctional . secreted neuronal proteins then activate nearby microglia that counter - react by secreting proinflammatory and potentially neurotoxic factors , fueling the viscous cycle that eventually leads to neuronal death . based on the combination of transgenic and viral models , and taking into account pathological and clinical data from human patients , we defend the thesis that neurodegeneration is not caused by a single defect but by  at least  dual actions ( figure 4 ) . the first is proposed to be intrinsically neuronal , for example , damage by accumulation of endogenous proteins in various molecular forms . in the case discussed here , protein tau is the cause in primary tauopathies . in ad , the upstream triggers are the amyloid peptides that accumulate because of mutations or other imposed defects in the proteolytic machinery that controls their formation and turnover . the second factor is also essential , and can be neuronal or microglial in origin , related to any type of stress the brain can experience or has to endure , that is , partial hypoxia , glucose overload or shortage , problems with lipid or cholesterol , or other essential metabolites . protein - aggregates , that is , plaques and tangles , are not the most essential in causing cognitive defects and dementia . the challenge to define the neurotoxic tau - species and its mode of action does nevertheless become not less complicated . the combination of transgenic and viral models illustrates eloquently the power of the approach as well as its weaknesses . we still have more modeling to do , taking into account the data and indications that are provided by ongoing clinical studies , because only the human patient can eventually confirm any hypothesis based on experimental models .